IκB Kinase-β (IKKβ) binding antibodies and methods of using same

ABSTRACT

The present invention provides an isolated nucleic acid molecules encoding IκB kinase (IKK) catalytic subunit polypeptides, which are associated with an IKK serine protein kinase that phosphorylates a protein (IκB) that inhibits the activity of the NF-κB transcription factor, vectors comprising such nucleic acid molecules and host cells containing such vectors. In addition, the invention provides nucleotide sequences that can bind to a nucleic acid molecule of the invention, such nucleotide sequences being useful as probes or as antisense molecules. The invention also provides isolated IKK catalytic subunits, which can phosphorylate an IκB protein, and peptide portions of such IKK subunit. In addition, the invention provides anti-IKK antibodies, which specifically bind to an IKK complex or an IKK catalytic subunit, and IKK-binding fragments of such antibodies. The invention further provides methods of substantially purifying an IKK complex, methods of identifying an agent that can alter the association of an IKK complex or an IKK catalytic subunit with a second protein, and methods of identifying proteins that can interact with an IKK complex or an IKK catalytic subunit.

This is a Divisional of U.S. application Ser. No. 09/796,872, filed onFeb. 28, 2001, now U.S. Pat. No. 6,689,575, which is a Divisional ofU.S. application Ser. No. 09/168,629, filed on Oct. 8, 1998, now U.S.Pat. No. 6,242,253, which is based on, and claims the benefit of, U.S.Provisional Application No. 60/061,470, filed Oct. 9, 1997, the entirecontents of which is herein incorporated by reference.

This invention was made with government support under grant numberCA50528 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to molecular biology andbiochemistry and more specifically to a protein kinase, IκB kinase,which is activated in response to environmental stresses andproinflammatory signals to phosphorylate inhibitors of the NF-κBtranscription factors and to methods of using the protein kinase.

2. Background Information

The induction of gene expression due to exposure of a cell to a specificstimulus is a tightly controlled process. Depending on the inducingstimulus, it can be critical to survival of the cell that one or moregenes be rapidly induced, such that the expressed gene product canmediate its effect. For example, an inflammatory response stimulated dueto an injury to or infection of a tissue results in rapid vasodilationin the area of the injury and infiltration of effector cells such asmacrophages. Vasodilation occurs within minutes of the response and isdue, in part, to the expression of cytokines in the injured region.

The rapid induction, for example, of an inflammatory response or animmune response, requires that the transcription factors involved inregulating such responses be present in the cell in a form that isamenable to rapid activation. Thus, upon exposure to an inducingstimulus, the response can occur quickly. If, on the other hand, suchtranscription factors were not already present in a cell in an inactivestate, the factors first would have to be synthesized upon exposure toan inducing stimulus, greatly reducing the speed with which a responsesuch as an inflammatory response could occur.

Regulation of the activity of transcription factors involved in suchrapid induction of gene expression can occur by various mechanisms. Forexample, in some cases, a transcription factor that exists in aninactive state in a cell can be activated by a post-translationalmodification such as phosphorylation on one or more serine, threonine ortyrosine residues. In addition, a transcription factor can be inactivedue to an association with a regulatory factor, which, upon exposure toan inducing stimulus, is released from the transcription factor, therebyactivating the transcription factor. Alternatively, an inactivetranscription factor may have to associate with a second protein inorder to have transcriptional activity.

Rarely, as in the case of glucocorticoids, the inducing stimulusinteracts directly with the inactive transcription factor, rendering itactive and resulting in the induction of gene expression. More often,however, an inducing stimulus initiates the induced response byinteracting with a specific receptor present on the cell membrane or byentering the cell and interacting with an intracellular protein.Furthermore, the signal generally is transmitted along a pathway, forexample, from the cell membrane to the nucleus, due to a series ofinteractions of proteins. Such signal transduction pathways allow forthe rapid transmission of an extracellular inducing stimulus such thatthe appropriate gene expression is rapidly induced.

Although the existence of signal transduction pathways has long beenrecognized and many of the cellular factors involved in such pathwayshave been described, the pathways responsible for the expression of manycritical responses, including the inflammatory response and immuneresponse, have not been completely defined. For example, it isrecognized that various inducing stimuli such as bacteria or virusesactivate common arms of the immune and inflammatory responses. However,differences in the gene products expressed also are observed, indicatingthat these stimuli share certain signal transduction pathways but alsoinduce other pathways unique to the inducing stimulus. Furthermore,since inducing agents such as bacteria or viruses initially stimulatedifferent signal transduction pathways, yet induce the expression ofcommon genes, some signal transduction pathways must converge at a pointsuch that the different pathways activate common transcription factors.

A clearer understanding of the proteins involved in such pathways canallow a description, for example, of the mechanism of action of a drugthat is known to interfere with the expression of genes regulated by aparticular pathway, but the target of which is not known. In addition,the understanding of such pathways can allow the identification of adefect in the pathway that is associated with a disease such as cancer.For example, the altered expression of cell adhesion molecules isassociated with the ability of a cancer cell to metastasize. However,the critical proteins involved in the signal transduction pathwayleading to expression of cell adhesion molecules have not beenidentified. Thus, a need exists to identify the proteins involved insignal transduction pathways, particularly those proteins present at theconvergence point of different initial pathways that result in theinduction, for example, of gene products involved in the inflammatoryand immune responses The present invention satisfies this need andprovides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules encodingfull length human serine protein kinases, designated IκB kinase (IKK)subunits IKKα and IKKβ. The disclosed IKK subunits share substantialsequence homology and are activated in response to proinflammatorysignals to phosphorylate proteins (IκB's) that inhibit the activity ofthe NF-κB transcription factor.

For example, the invention provides a nucleic acid molecule having thenucleotide sequence shown as SEQ ID NO: 1, which encodes a cytokineinducible IκB kinase subunit designated IKKα, particularly the sequenceshown as nucleotides −35 to 92 in SEQ ID NO: 1, and nucleic acidmolecules encoding the amino acid sequence shown as SEQ ID NO: 2, aswell as nucleotide sequences complementary thereto. In addition, theinvention provides a nucleic acid molecule having the nucleotidesequence shown as SEQ ID NO: 14, which encodes a second cytokineinducible IκB kinase subunit, designated IKKβ, and nucleic acidmolecules encoding the amino acid sequence shown as SEQ ID NO: 15, aswell nucleotide sequences complementary thereto. The invention alsoprovides vectors comprising the nucleic acid molecules of the inventionand host cells containing such vectors.

In addition, the invention provides nucleotide sequences that bind to anucleic acid molecule of the invention, including to nucleotides −35 to92 as shown in SEQ ID NO: 1. Such nucleotide sequences of the inventionare useful as probes, which can be used to identify the presence of anucleic acid molecule encoding an IKK subunit in a sample, and asantisense molecules, which can be used to inhibit the expression of anucleic acid molecule encoding an IKK subunit.

The present invention also provides isolated full length human IKKsubunits, which can phosphorylate an IκB protein. For example, theinvention provides an IKKα polypeptide having the amino acid sequenceshown as SEQ ID NO: 2, particularly the amino acid sequence comprisingamino acids 1 to 31 at the N-terminus of the polypeptide of SEQ ID NO:2. In addition, the invention provides an IKKβ polypeptide having theamino acid sequence shown as SEQ ID NO: 15. The invention also providespeptide portions of an IKK subunit, including, for example, peptideportions comprising one or more contiguous amino acids of the N-terminalamino acids shown as residues 1 to 31 in SEQ ID NO: 2. A peptide portionof an IKK subunit can comprise the kinase domain of the IKK subunit orcan comprise a peptide useful for eliciting production of an antibodythat specifically binds to an IκB kinase or to the IKK subunit.Accordingly, the invention also provides anti-IKK antibodies thatspecifically bind to an IKK complex comprising an IKK subunit,particularly to the IKK subunit, for example, to an epitope comprisingat least one of the amino acids shown as residues 1 to 31 of SEQ ID NO:2, and also provides IKK subunit-binding fragments of such antibodies.In addition, the invention provides cell lines producing anti-IKKantibodies or IKK-binding fragments thereof.

The invention also provides isolated IκB kinase complexes. As disclosedherein, an IKK complex can have an apparent molecular mass of about 900kDa or about 300 kDa. An IKK complex is characterized, in part, in thatit comprises an IKKα subunit, an IKKβ subunit, or both and canphosphorylate an IκB protein.

The present invention further provides methods for isolating an IKKcomplex or an IKK subunit, as well as methods of identifying an agentthat can alter the association of an IKK complex or an IKK subunit witha second protein that associates with the IKK in vitro or in vivo. Sucha second protein can be, for example, another IKK subunit; an IκBprotein, which is a substrate for IKK activity and is involved in asignal transduction pathway that results in the regulated expression ofa gene; a protein that is upstream of the IκB kinase in a signaltransduction pathway and regulates IKK activity; or a protein that actsas a regulatory subunit of the IκB kinase or of an IKK subunit and isnecessary for full activation of the IKK complex. An agent that altersthe association of an IKK subunit with a second protein can be, forexample, a peptide, a polypeptide, a peptidomimetic or a small organicmolecule. Such agents can be useful for modulating the level ofphosphorylation of IκB in a cell, thereby modulating the activity ofNF-κB in the cell and the expression of a gene regulated by NF-κB.

The invention also provides methods of identifying proteins that caninteract with an IκB kinase, including with an IKK subunit, suchproteins which can be a downstream effector of the IKK such as a memberof the IκB family of proteins or an upstream activator or a regulatorysubunit of an IKK. Such proteins that interact with an IKK complex orthe IKK subunit can be isolated, for example, by coprecipitation withthe IKK or by using the IKK subunit as a ligand, and can be involved,for example, in tissue specific regulation of NF-κB activation andconsequent tissue specific gene expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO: 1; lower case letter) anddeduced amino acid sequence (SEQ ID NO: 2; upper case letters) of fulllength human IKKα subunit of an IKK complex. Nucleotide positions areindicated to the right and left of the sequence; the “A” of the ATGencoding the initiator methionine is shown as position 1. Underlinedamino acid residues indicate the peptide portions of the protein(“peptide 1” and “peptide 2”) that were sequenced and used to designoligonucleotide probes. The asterisk indicates the sequence encoding theSTOP codon.

FIG. 2 shows a nucleotide sequence (SEQ ID NO: 14) encoding a fulllength IKKβ polypeptide (see FIG. 3). Numbers to the left and right ofthe sequence indicate nucleotide position number. The initiator ATGcodon is present at nucleotides 36-38 and the first stop codon (TGA) ispresent at nucleotides 2304-2306.

FIG. 3 shows an alignment of the deduced amino acid sequences of IKKα(“α”, SEQ ID NO: 2) and IKKβ (“β”, SEQ ID NO: 15). Numbers to the rightof the sequences indicate the respective amino acid positions.Underlined amino acid residues indicate peptide portions of the IKKβsubunit that were sequenced and used to search an EST database (seeExample III). Vertical bars between amino acid residues indicateidentical amino acids; two dots between amino acid residues indicatesvery similar amino acids (e.g., Glu and Asp; Arg and Lys) and one dotbetween amino acid residues indicates a lesser degree of similarity. Adot within an amino acid sequence indicates a space introduced tomaintain sequence homology. The kinase domains in the N-terminal half ofthe sequences and helix-loop-helix domains in the C-terminal half of thesequences are bracketed and the leucine residues involved in the leucinezippers are indicated by the filled circles above the IKKα sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acid molecules encodingpolypeptide subunits of human serine protein kinase complex, the IκBkinase (IKK), which is activated in response to proinflammatory signalsand phosphorylates proteins (IκB's) that bind to and inhibit theactivity of NF-κB transcription factors. For example, the inventionprovides an isolated nucleic acid molecule (SEQ ID NO: 1) encoding afull length human IKKα subunit having the amino acid sequence shown asSEQ ID NO: 2 (FIG. 1). In addition, the invention provides an isolatednucleic acid molecule (SEQ ID NO: 14; FIG. 2) encoding a full lengthhuman IKKβ subunit having the amino acid sequence shown as SEQ ID NO: 15(FIG. 3).

As used herein, the term “isolated,” when used in reference to a nucleicacid molecule of the invention, means that the nucleic acid molecule isrelatively free from contaminating lipids, proteins, nucleic acids orother cellular material normally associated with a nucleic acid moleculein a cell. An isolated nucleic acid molecule of the invention can beobtained, for example, by chemical synthesis of the nucleotide sequenceshown as SEQ ID NO: 1 or SEQ ID NO: 14 or by cloning the molecule usingmethods such as those disclosed in Examples II and III. In general, anisolated nucleic acid molecule comprises at least about 30% of a samplecontaining the nucleic acid molecule, and generally comprises about 50%or 70% or 90% of a sample, preferably 95% or 98% of the sample. Such anisolated nucleic acid molecule can be identified by comparing, forexample, a sample containing the isolated nucleic acid molecule with thematerial from which the sample originally was obtained. Thus, anisolated nucleic acid molecule can be identified, for example, bycomparing the relative amount of the nucleic acid molecule in fractionof a cell lysate obtained following gel electrophoresis with therelative amount of the nucleic acid molecule in the cell, itself.

IKKα and IKKβ have been designated IKK subunits because they arecomponents of an approximately 900 kDa complex having IκB kinase (IKK)activity and because they share substantial nucleotide and amino acidsequence homology. As disclosed herein, IKKα and IKKβ are relatedmembers of a family of IKK catalytic subunits (see FIG. 3). The 900 kDaIκB kinase complex can be isolated in a single step, for example, byimmunoprecipitation using an antibody specific for an IKK subunit or byusing metal ion chelation chromatography methods (see Example IV). A 300kDa IKK complex also can be isolated as disclosed herein and has kinaseactivity for an IκB substrate (see Example III).

Nucleic acid molecules related to SEQ ID NO: 1 previously have beendescribed (Connelly and Marcu, Cell. Mol. Biol. Res. 41:537-549 (1995),which is incorporated herein by reference). For example, Connelly andMarcu describe a 3466 base pair (bp) nucleic acid molecule (GenBankAccession #U12473; Locus MMU 12473), which is incorporated herein byreference), which encodes a full length mouse polypeptide having anapparent molecular mass of 85 kiloDaltons (kDa) and designated CHUK. A2146 bp nucleic acid molecule (GenBank Accession #U22512; Locus HSU22512), which is incorporated herein by reference), which encodes aportion of the polypeptide shown in SEQ ID NO: 2 also was described.However, the amino acid sequence deduced from #U22512 lacks amino acids1 to 31 as shown in SEQ ID NO: 2 and, therefore, is not a full lengthprotein. In addition, several nucleotide differences occur in SEQ ID NO:1 as compared to the sequence of #U22512, including nucleotide changesthat encode different amino acids at positions 543, 604, 679, 680, 684and 685 of SEQ ID NO: 2; silent nucleotide changes also occur at codons665 and 678. The polypeptides encoded by the nucleotide sequences ofGenBank Accession #U12473 and #U22512 share about 95% identity at theamino acid level and are substantially similar to that shown in SEQ IDNO: 2. No function has been demonstrated for the polypeptides describedby Connelly and Marcu, although Regnier et al. (Cell 90:373-383 (1997))recently have confirmed that human CHUK corresponds to IKKα, asdisclosed herein.

A nucleic acid molecule of the invention is exemplified by thenucleotide sequences shown as SEQ ID NO: 1, which encodes a full lengthhuman IKKα (SEQ ID NO: 2; FIG. 1), the activity of which is stimulatedby a cytokine or other proinflammatory signal, and as SEQ ID NO: 14,which encodes a full length IKKβ (SEQ ID NO: 15). Due to the degeneracyof the genetic code and in view of the disclosed amino acid sequence ofa full length human IKKα (SEQ ID NO: 2) and of the IKKβ (SEQ ID NO: 15),additional nucleic acid molecules of the invention would be well knownto those skilled in the art. Such nucleic acid molecules, respectively,have a nucleotide sequence that is different from SEQ ID NO: 1 but,nevertheless, encodes the amino acid sequence shown as SEQ ID NO: 2, orhave a nucleotide sequence that is different from SEQ ID NO: 14 but,nevertheless, encodes the amino acid sequence shown as SEQ ID NO: 15.Thus, the invention provides a nucleic acid molecule comprising anucleotide sequence encoding the amino acid sequence of a full lengthhuman IKKα as shown in SEQ ID NO: 2 or of IKKβ as shown in SEQ ID NO:15.

As used herein, reference to “a nucleic acid molecule encoding an IKKsubunit” indicates 1) the polynucleotide sequence of one strand of adouble stranded DNA molecule comprising the nucleotide sequence thatcodes for the IKK subunit and can be transcribed into an RNA thatencodes the IKK subunit, or 2) an RNA molecule, which can be translatedinto an IKK subunit. It is recognized that a double stranded DNAmolecule also comprises a second polynucleotide strand that iscomplementary to the coding strand and that the disclosure of apolynucleotide sequence comprising a coding sequence necessarilydiscloses the complementary polynucleotide sequence. Accordingly, theinvention provides polynucleotide sequences, including, for example,polydeoxyribonucleotide or polyribonucleotide sequences that arecomplementary to the nucleotide sequence shown as SEQ ID NO: 1 or as-SEQID NO: 14, or to a nucleic acid molecule encoding an IKK catalyticsubunit having the amino acid sequence shown as SEQ ID NO: 2 or as SEQID NO: 15, respectively.

As used herein, the term “polynucleotide” is used in its broadest senseto mean two or more nucleotides or nucleotide analogs linked by acovalent bond. The term “oligonucleotide” also is used herein to meantwo or more nucleotides or nucleotide analogs linked by a covalent bond,although those in the art will recognize that oligonucleotides generallyare less than about fifty nucleotides in length and, therefore, are asubset within the broader meaning of the term “polynucleotide.”

In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotidealso can comprise nucleotide analogs, including non-naturally occurringsynthetic nucleotides or modified naturally occurring nucleotides. Suchnucleotide analogs are well known in the art and commercially available,as are polynucleotides containing such nucleotide analogs (Lin et al.,Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry34:11363-11372 (1995); Pagratis et al., Nature Biotechnol. 15:68-73(1997)). The covalent bond linking the nucleotides of a polynucleotidegenerally is a phosphodiester bond. However, the covalent bond also canbe any of numerous other bonds, including a thiodiester bond, aphosphorothioate bond, a peptide-like bond or any other bond known tothose in the art as useful for linking nucleotides to produce syntheticpolynucleotides (see, for example, Tam et al., Nucl. Acids Res.22:977-986 (1994); Ecker and Crooke, BioTechnology 13:351360 (1995)).

Where it is desired to synthesize a polynucleotide of the invention, theartisan will know that the selection of particular nucleotides ornucleotide analogs and the covalent bond used to link the nucleotideswill depend, in part, on the purpose for which the polynucleotide isprepared. For example, where a polynucleotide will be exposed to anenvironment containing substantial nuclease activity, the artisan willselect nucleotide analogs or covalent bonds that are relativelyresistant to the nucleases. A polynucleotide comprising naturallyoccurring nucleotides and phosphodiester bonds can be chemicallysynthesized or can be produced using recombinant DNA methods, using anappropriate polynucleotide as a template. In comparison, apolynucleotide comprising nucleotide analogs or covalent bonds otherthan phosphodiester bonds generally will be chemically synthesized,although an enzyme such as T7 polymerase can incorporate certain typesof nucleotide analogs and, therefore, can be used to produce such apolynucleotide recombinantly from an appropriate template (Jellinek etal., supra, 1995).

The invention also provides nucleotide sequences that can specificallyhybridize to a nucleic acid molecule of the invention. Such hybridizingnucleotide sequences are useful, for example, as probes, which canhybridize to a nucleic acid molecule encoding an IKK catalytic subunitand allow the identification of the nucleic acid molecule in a sample. Anucleotide sequence of the invention is characterized, in part, in thatit is at least nine nucleotides in length, such sequences beingparticularly useful as primers for the polymerase chain reaction (PCR),and can be at least fourteen nucleotides in length or, if desired, atleast seventeen nucleotides in length, such nucleotide sequences beingparticularly useful as hybridization probes, although such sequencesalso can be used for PCR. A nucleotide sequence of the invention cancomprise at least six nucleotides 5′ to nucleotide position 92 as shownin SEQ ID NO: 1 (FIG. 1), preferably at least nine nucleotides 5′ toposition 92, or more as desired, where SEQ ID NO: 1 is shown in theconventional manner from the 5′-terminus (FIG. 1; upper left) to the3′-terminus. Such nucleotide sequences of the invention are particularlyuseful in methods of diagnosing a pathology, for example, a humandisease, characterized by aberrant IKK activity. For convenience, suchnucleotide sequences can comprise a kit, which can be made commerciallyavailable and can provide a standardized diagnostic assay.

A nucleic acid molecule encoding an IKKα such as the nucleotide sequenceshown in SEQ ID NO: 1 diverges from the sequence encoding the mousehomolog (GenBank Accession #U12473) in the region encoding amino acid30. Thus, a nucleotide sequence comprising nucleotides 88 to 90 as shownin SEQ ID NO: 1, which encodes amino acid 30 of human IKKα, can beparticularly useful, for example, for identifying the presence of anucleic acid molecule encoding a human IKKα in a sample. Furthermore,based on a comparison of SEQ ID NO: 1 with SEQ ID NO: 14, the skilledartisan readily can select nucleotide sequences that can hybridize witha nucleic acid molecule encoding a human IKKα or a human IKKβ or both bydesigning the sequence to contain conserved or non-conserved nucleotidesequences, as desired. For example, selection of a nucleotide sequencethat is highly conserved among SEQ ID NO: 1 and SEQ ID NO: 14 can allowthe identification of related members of the IKK subunit family ofproteins. In comparison, selection of a nucleotide sequence that ispresent, for example, in SEQ ID NO: 14, but that is not present in SEQID NO: 1 or that shares only minimal homology can allow identificationof the expression of SEQ ID NO: 14 in a cell, irrespective of whetherSEQ ID NO: 1 also is expressed in the cell. It should be recognized,however, that a nucleotide sequence of the invention readily isidentifiable in comparison to GenBank Accession #U12473 or #U22512 inthat a nucleotide sequence of the invention is not the nucleotidesequence of GenBank Accession #U12473 or #U22512.

A nucleotide sequence of the invention can comprise a portion of acoding sequence of a nucleic acid molecule encoding an IKK subunit or ofa sequence complementary thereto, depending on the purpose for which thenucleotide sequence is to be used. In addition, a mixture of a codingsequence and its complementary sequence can be prepared and, if desired,can be allowed to anneal to produce double stranded molecules.

The invention also provides antisense nucleic acid molecules, which arecomplementary to a nucleic acid molecule encoding an IKK subunit and canbind to and inhibit the expression of the nucleic acid molecule. Asdisclosed herein, expression of an antisense molecule complementary tothe nucleotide sequence shown in SEQ ID NO: 1 inhibited the cytokineinducible expression of an NF-κB dependent reporter gene in a cell(Example II.B.). Thus, an antisense molecule of the invention can beuseful for decreasing IKK activity in a cell, thereby reducing orinhibiting the level of NF-κB mediated gene expression. Theseexperiments were performed twenty-four hours after the cells weretransfected (Example II.B.). Expression of the antisense molecule in thecell also resulted in a decreased level of IKKα activity as compared tovector transfected control cells, indicating that the IKKβ has arelatively short half life. Antisense nucleic acid molecules specificfor IKKα or for IKKβ or for both can be designed based on the criteriadiscussed above for the selection of hybridizing nucleotide sequences.

An antisense nucleic acid molecule of the invention can comprise asequence complementary to the entire coding sequence of an IKK catalyticsubunit such as a sequence complementary to SEQ ID NO: 1 or SEQ ID NO:14, provided the antisense sequence is not complementary in its entiretyto the sequences of GenBank Accession #U12473 or #U22512. In addition, anucleotide sequence complementary to a portion of a nucleic acidmolecule encoding an IKK subunit can be useful as an antisense molecule,particularly a nucleotide sequence complementary to nucleotides −35 to92 of SEQ ID NO: 1 or, for example, a nucleotide sequence comprising atleast 9 nucleotides on each side of the ATG encoding the initiatormethionine (complementary to positions −9 to 12 of SEQ ID NO: 1) or, ifdesired, at least 17 nucleotides on each side of the ATG codon(complementary to positions −17 to 20 of SEQ ID NO: 1), or to thecorresponding sequences of SEQ ID NO: 14.

Antisense methods involve introducing the nucleic acid molecule, whichis complementary to and can hybridize to the target nucleic acidmolecule, into a cell. An antisense nucleic acid molecule can be achemically synthesized polynucleotide, which can be introduced into thetarget cells by methods of transfection, or can be expressed from aplasmid or viral vector, which can be introduced into the cell andstably or transiently expressed using well known methods (see, forexample, Sambrook et al., Molecular Cloning: A laboratory manual (ColdSpring Harbor Laboratory Press 1989); Ausubel et al., Current Protocolsin Molecular Biology (Green Publ., NY 1989), each of which isincorporated herein by reference). One in the art would know that theability of an antisense (or other hybridizing) nucleotide sequence tospecifically hybridize to the target nucleic acid sequence depends, forexample, on the degree of complementarity shared between the sequences,the GC content of the hybridizing molecules, and the length of theantisense nucleic acid sequence, which can be at least ten nucleotidesin length, generally at least thirty nucleotides in length or at leastfifty nucleotides in length, and can be up to the full length of anucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 14 or a nucleotidesequence encoding an IKK subunit as shown in SEQ ID NO: 2 or in SEQ IDNO: 15 (see Sambrook et al., supra, 1989).

The invention also provides vectors comprising a nucleic acid moleculeof the invention and host cells, which are appropriate for maintainingsuch vectors. Vectors, which can be cloning vectors or expressionvectors, are well known in the art and commercially available. Anexpression vector comprising a nucleic acid molecule of the invention,which can encode an IKK-α or can be an antisense molecule, can be usedto express the nucleic acid molecule in a cell.

In general, an expression vector contains the expression elementsnecessary to achieve, for example, sustained transcription of thenucleic acid molecule, although such elements also can be inherent tothe nucleic acid molecule cloned into the vector. In particular, anexpression vector contains or encodes a promoter sequence, which canprovide constitutive or, if desired, inducible expression of a clonednucleic acid sequence, a poly-A recognition sequence, and a ribosomerecognition site, and can contain other regulatory elements such as anenhancer, which can be tissue specific. The vector also containselements required for replication in a procaryotic or eukaryotic hostsystem or both, as desired. Such vectors, which include plasmid vectorsand viral vectors such as bacteriophage, baculovirus, retrovirus,lentivirus, adenovirus, vaccinia virus, semliki forest virus andadeno-associated virus vectors, are well known and can be purchased froma commercial source (Promega, Madison Wis.; Stratagene, La Jolla Calif.;GIBCO/BRL, Gaithersburg Md.) or can be constructed by one skilled in theart (see, for example, Meth. Enzymol., Vol. 185, D. V. Goeddel, ed.(Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64 (1994);Flotte, J. Bioenerg. Biomemb. 25:37-42 (1993); Kirshenbaum et al., J.Clin. Invest 92:381-387 (1993), which is incorporated herein byreference).

A nucleic acid molecule, including a vector, can be introduced into acell by any of a variety of methods known in the art (Sambrook et al.,supra, 1989, and in Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1994), which isincorporated herein by reference). Such methods include, for example,transfection, lipofection, microinjection, electroporation and infectionwith recombinant vectors or the use of liposomes.

Introduction of a nucleic acid molecule by infection with a viral vectoris particularly advantageous in that it can efficiently introduce thenucleic acid molecule into a cell ex vivo or in vivo. Moreover, virusesare very specialized and typically infect and propagate in specific celltypes. Thus, their natural specificity can be used to target the nucleicacid molecule contained in the vector to specific cell types. Forexample, a vector based on HIV-1 can be used to target an antisense IKKsubunit molecule to HIV-1 infected cells, thereby reducing thephosphorylation of IκB, which can decrease the high level ofconstitutive NF-κB activity present in HIV-1 infected cells. Viral ornon-viral vectors also can be modified with specific receptors orligands to alter target specificity through receptor mediated events.

A nucleic acid molecule also can be introduced into a cell using methodsthat do not require the initial introduction of the nucleic acidmolecule into a vector. For example, a nucleic acid molecule encoding anIKK catalytic subunit can be introduced into a cell using a cationicliposomes, which also can be modified with specific receptors or ligandsas described above (Morishita et al., J. Clin. Invest., 91:2580-2585(1993), which is incorporated herein by reference; see, also, Nabel etal., supra, 1993)). In addition, a nucleic acid molecule can beintroduced into a cell using, for example, adenovirus-polylysine DNAcomplexes (see, for example, Michael et al., J. Biol. Chem.,268:6866-6869 (1993), which is incorporated herein by reference). Othermethods of introducing a nucleic acid molecule into a cell such that theencoded IKK subunit or antisense nucleic acid molecule can be expressedare well known (see, for example, Goeddel, supra, 1990).

Selectable marker genes encoding, for example, a polypeptide conferringneomycin resistance (Neo^(R)) also are readily available and, whenlinked to a nucleic acid molecule of the invention or incorporated intoa vector containing the nucleic acid molecule, allows for the selectionof cells that have incorporated the nucleic acid molecule. Otherselectable markers such as that conferring hygromycin, puromycin orZEOCIN (Invitrogen) resistance are known to those in the art of genetransfer can be used to identify cells containing the nucleic acidmolecule, including the selectable marker gene.

A “suicide” gene also can be incorporated into a vector so as to allowfor selective inducible killing of a cell containing the gene. A genesuch as the herpes simplex virus thymidine kinase gene (TK) can be usedas a suicide gene to provide for inducible destruction of such cells.For example, where it is desired to terminate the expression of anintroduced nucleic acid molecule encoding IKK or an antisense IKKsubunit molecule in cells containing the nucleic acid molecule, thecells can be exposed to a drug such as acyclovir or gancyclovir, whichcan be administered to an individual.

Numerous methods are available for transferring nucleic acid moleculesinto cultured cells, including the methods described above. In addition,a useful method can be similar to that employed in previous human genetransfer studies, where tumor infiltrating lymphocytes (TILs) weremodified by retroviral gene transduction and administered to cancerpatients (Rosenberg et al., New Engl. J. Med. 323:570-578 (1990)). Inthat Phase I safety study of retroviral mediated gene transfer, TILswere genetically modified to express the Neomycin resistance (Neo^(R))gene. Following intravenous infusion, polymerase chain reaction analysesconsistently found genetically modified cells in the circulation for aslong as two months after administration. No infectious retroviruses wereidentified in these patients and no side effects due to gene transferwere noted in any patients. These retroviral vectors have been alteredto prevent viral replication by the deletion of viral gag, pol and envgenes. Such a method can also be used ex vivo to transduce cells takenfrom a subject (see Anderson et al., U.S. Pat. No. 5,399,346, issuedMar. 21, 1995, which is incorporated herein by reference).

When retroviruses are used for gene transfer, replication competentretroviruses theoretically can develop due to recombination ofretroviral vector and viral gene sequences in the packaging cell lineutilized to produce the retroviral vector. Packaging cell lines in whichthe production of replication competent virus by recombination has beenreduced or eliminated can be used to minimize the likelihood that areplication competent retrovirus will be produced. Hence, all retroviralvector supernatants used to infect cells will be screened forreplication competent virus by standard assays such as PCR and reversetranscriptase assays.

To function properly, a cell requires the precise regulation ofexpression of nearly all genes. Such gene regulation is accomplished byactivation or repression of transcription by various transcriptionfactors, which interact directly with regulatory sequences on nuclearDNA. The ability of transcription factors to bind DNA or activate orrepress transcription is regulated in response to external stimuli. Inthe case of the transcription factor NF-κB, critical factors involved inthe signaling pathway mediating its activation have not been identified(Verma, et al., Genes Devel. 9:2723-2735 (1995); Baeuerle and Baltimore,Cell 87:13-20 (1996)).

NF-κB is a member of the Rel family of transcription factors, which arepresent in most if not all animal cells (Thanos and Maniatis, Cell80:629-532 (1995)). Rel proteins, which include, for example, RelA(p65), c-Rel, p50, p52 and the Drosophila dorsal and Dif gene products,are characterized by region of about 300 amino acids sharingapproximately 35% to 61% homology (“Rel homology domain”). The Relhomology domain includes DNA binding and dimerization domains and anuclear localization signal. Rel proteins are grouped into one of twoclasses, depending on whether the protein also contains atranscriptional activation domain (Siebenlist et al., Ann. Rev. CellBiol. 10:405-455 (1994)).

Rel proteins can from homodimers or heterodimers, which can betranscriptionally activating depending on the presence of atransactivation domain. The most common Rel/NF-κB dimer, which isdesignated “NF-κB,” is a p50/p65 heterodimer that can activatetranscription of genes containing the appropriate κB binding sites.p50/p65 NF-κB is present in most cell types and is considered theprototype of the Rel/NF-κB family of transcription factors. Differentdimers vary in their binding to different κKB elements, kinetics ofnuclear translocation and levels of expression in a tissue (Siebenlistet al., supra, 1994). As used herein, the term “Rel/NF-κB” is used torefer generally to the Rel family of transcription factors, and the term“NF-κB” is used to refer specifically to the Rel/NF-κB factor consistingof a p50/p65 heterodimer.

NF-κB originally was identified by its ability to bind a specific DNAsequence present in the immunoglobulin κ light chain gene enhancer, the“κB element” (Sen and Baltimore, Cell 46:705-709 (1986)). The κB elementhas been identified in numerous cellular and viral promotors, includingpromotors present in human immunodeficiency virus-1 (HIV-1);immunoglobulin superfamily genes such as the MHC class 1 (H-2κ) gene;cytokine genes such as the tumor necrosis factor α (TNFα),interleukin-1β (IL-1β), IL-2, IL-6 and the granulocyte-macrophage colonystimulating factor (GM-CSF) gene; chemokine genes such as RANTES andIL-8; and cell adhesion protein genes such as E-selectin. The κB elementexhibits dyad symmetry and each half site of the element likely is boundby one subunit of an NF-κB dimer.

In the absence of an appropriate signaling stimulus, a Rel/NF-κB ismaintained in the cytoplasm in an inactive form complexed with an IκBprotein. Rel/NF-κB transcriptional activity is induced by numerouspathogenic events or stresses, including cytokines, chemokines, virusesand viral products, double stranded RNA, bacteria and bacterial productssuch as lipopolysaccharide (LPS) and toxic shock syndrome toxin-1,mitogens such as phorbol esters, physical and oxidative stresses, andchemical agents such as okadaic acid and cycloheximide (Thanos andManiatis, supra, 1995; Siebenlist et al., supra, 1994). Significantly,the expression of genes encoding agents such as TNFα, IL-1, IL-6,interferon-β and various chemokines, which induce NF-κB activity, are,themselves, induced by NF-κB, resulting in amplification of their signalby a positive, self-regulatory loop (Siebenlist et al., supra, 1994).Phorbol esters, which activate T cells, also activate NF-κB andimmunosuppressants such as cyclosporin A inhibit activation of T cellsthrough T cell receptor mediated signals (Baldwin, Ann. Rev. Immunol.14:649-681 (1996), which is incorporated herein by reference).

Regulation of specific genes by NF-κB can require interaction of NF-κBwith one or more other DNA binding proteins. For example, expression ofE-selectin requires an interaction of NF-κB, the bZIP protein ATF-2 andHMG-I(Y), and expression of the IL-2 receptor a gene requires aninteraction of NF-κB, HMG-I(Y) and the ets-like protein, ELF-1 (Baldwin,supra, 1996).

The numerous agents that induce activation of NF-κB likely act throughvarious converging signal transduction pathways, including pathwaysinvolving activation of protein kinase C, Raf kinase and tyrosinekinases. The ability of antioxidants to inhibit NF-κB activation byvarious inducing agents suggests that reactive oxygen species are aconverging point of such pathways (Siebenlist et al., supra, 1994).

Upon activation by an appropriate inducing agent, a Rel/NF-κB dimer istranslocated into the nucleus, where it can activate gene transcription.The subcellular localization of a Rel/NF-κB is controlled by specificinhibitory proteins (“inhibitors of Rel/NF-κB” or “IκB's”), whichnoncovalently bind the Rel/NF-κB and mask its nuclear localizationsignal (NLS), thereby preventing nuclear uptake. Various IκB's,including, for example, IκBα, IκBβ, Bcl-3 and the Drosophila cactus geneproduct, have been identified (Baeuerle and Baltimore, supra, 1996). Inaddition, Rel precursor proteins, such as p105 and p100, which areprecursors of p50 and p52, respectively, function as IκB's (Siebenlistet al., supra, 1994). IκBα and IκBβ are expressed in most cell types andgenerally bind p65- and c-Rel-containing Rel/NF-κB dimers. Other IκB'sappear to be expressed in a tissue specific manner (Thompson et al.,Cell 80:573-582 (1995)).

IκB proteins are characterized by the presence of 5 to 8 ankyrin repeatdomains, each about 30 amino acids, and a C-terminal PEST domain. Forexample, IκBα contains a 70 amino acid N-terminal domain, a 205 aminoacid internal domain containing the ankyrin repeats, and a 42 amino acidC-terminal domain containing the PEST domain (Baldwin, supra, 1996).Although IκB proteins interact through their ankyrin repeats with theRel homology domain of Rel/NF-κB dimers, binding of particular IκBproteins with particular Rel/NF-κB proteins appears to be relativelyspecific. For example, IκBα and IκBβ associate primarily with RelA- andc-Rel-containing Rel/NF-κB dimers, thereby blocking their nuclearlocalization signal. The binding of an IκB to NF-κB also interferes withthe ability of NF-κB to bind DNA. However, whereas IκBα isphosphorylated following exposure of cells to tumor necrosis factor(TNF), IL-1, bacterial lipopolysaccharide (LPS) or phorbol esters, IκBβis phosphorylated in certain cell types only in response to LPS or IL-1(Baldwin, supra, 1996). However, in other cell types, IκBβ isphosphorylated in response to the same signals that induce IκBα,although with slower kinetics than IκBα (DiDonato et al., Mol. Cell.Biol. 16:1295-1304 (1996), which is incorporated herein by reference).

Formation of a complex between an IκB protein and a Rel protein is dueto an interaction of the ankyrin domains with a Rel homology domain(Baeuerle and Baltimore, supra, 1996). Upon exposure to an appropriatestimulus, the IκB portion of the complex is rapidly degraded and theRel/NF-κB portion becomes free to translocate to the cell nucleus. Thus,activation of a Rel/NF-κB does not require de novo protein synthesisand, therefore, occurs extremely rapidly. Consequently, activation ofgene expression due to a Rel/NF-κB can be exceptionally rapid andprovides an effective means to respond to an external stimulus. Such arapid response of Rel/NF-κB transcription factors is particularlyimportant since these factors are involved in the regulation of genesinvolved in the immune, inflammatory and acute phase responses,including responses to viral and bacterial infections and to variousstresses.

Upon exposure of a cell to an appropriate inducing agent, IκBα, forexample, is phosphorylated at serine residue 32 (Ser-32) and Ser-36(Haskill et al., Cell 65:1281-1289 (1991)). Phosphorylation of IκBαtriggers its rapid ubiquitination, which results in proteasome-mediateddegradation of the inhibitor and translocation of active NF-κB to thenucleus (Brown et al., Science 267:1485-1488 (1995); Scherer et al.,Proc. Natl. Acad. Sci., USA. 92:11259-11263 (1995); DiDonato et al.,supra, 1996; DiDonato et al., Mol. Cell. Biol. 15:1302-1311 (1995);Baldi et al., J. Biol. Chem. 271:376-379 (1996)). The same mechanismalso accounts for IκBβ degradation (DiDonato et al., supra, 1996).

Rel/NF-κB activation can be transient or persistent, depending on theinducing agent and the IκB that is phosphorylated. For example, exposureof a cell to particular cytokines induces IκBα phosphorylation anddegradation, resulting in NF-κB activation, which induces the expressionof various genes, including the gene encoding IκBα. The newly expressedIκBα then binds to NF-κB in the nucleus, resulting in its export to thecytoplasm and inactivation and, therefore, a transient NF-κB mediatedresponse. In comparison, bacterial LPS induces IκBβ phosphorylation,resulting in NF-κB activation. However, the IκBβ gene is not induced byNF-κB and, as a result, activation of NF-κB is more persistent (Thompsonet al., supra, 1995).

A constitutively active multisubunit kinase of approximately 700 kDaphosphorylates IκBα at Ser-32 and Ser-36 and, in some cases, requirespolyubiquitination for activity (Chen et al., Cell 84:853-862 (1996);Lee et al., Cell 88:213-222 (1997)). The mitogen-activated proteinkinase/ERK kinase kinase-1 (MEKK1) phosphorylates several proteins thatcopurify with this complex and have molecular weights of approximately105 kDa, 64 kDa and 54 kDa; three other copurifying proteins havingmolecular weights of about 200 kDa, 180 kDa and 120 kDa arephosphorylated in the absence of MEKK1 (Lee et al., supra, 1997).However, a catalytically inactive MEKKL mutant, which can block TNFαmediated activation of the jun kinase, does not block NF-κB activation(Liu et al., Cell 87:565-576 (1996)).

Overexpression of MEKK1 also induces the site-specific phosphorylationof IκBα in vivo and can directly activate IκBα in vitro by anubiquitin-independent mechanism. However, MEKK1 did not phosphorylateIκBα at Ser-32 and Ser-36 in the in vitro experiments, indicating thatit is not an IκBα kinase, but may act upstream of IκBα kinase in asignal transduction pathway (Lee et al., supra, 1997).

In addition to the above described ubiquitin dependent kinase 700 kDacomplex, an ubiquitin independent 700 kDa complex, as well as anubiquitin independent 300 kDa kinase complex phosphorylates IκBα Ser-32and Ser-36, but not a mutant containing threonines substituted for theseserines (Baeuerle and Baltimore, supra, 1996). The specific polypeptidesresponsible for the IκB kinase activity of these complexes have not beendescribed.

A double stranded RNA-dependent protein kinase (PKR) that phosphorylatesIκBα in vitro has been described (Kumar et al., Proc. Natl. Acad. Sci.,USA 91:6288-6292 (1994)). Moreover, an antisense PKR DNA moleculeprevented NF-κB activation by double stranded RNA, but did not preventNF-κB activation by TNFα (Maran et al., Science 265:789-792 (1995)).Casein kinase II (CKII) also can interact with and phosphorylate IκBα,although weakly as compared to CKII phosphorylation of casein, and theSer-32 and Ser-36 residues in IκBα represent CKII phosphorylation sites(Roulston et al., supra, 1995). However, all of the inducers of NF-κBactivity do not stimulate these protein kinases to phosphorylate IκB,indicating that, if they are involved in NF-κB activation, thesekinases, like MEKK1, operate upstream of the IκB kinase. Thus, a rapidlystimulated IκB kinase that directly phosphorylates IκBα on Ser-32 andSer-36 and results in activation of NF-κB has not been identified.

A putative serine-threonine protein kinase has been identified in mousecells by probing for nucleic acid molecules that encode proteinscontaining a consensus helix-loop-helix domain, which is involved inprotein-protein interactions (Connelly and Marcu, supra, 1995). Thisputative kinase, which is ubiquitously expressed in various establishedcell lines, but differentially expressed in normal mouse tissues, wasnamed CHUK (conserved helix-loop-helix ubiquitous kinase; GenBankAccession #U12473). In addition, a nucleic acid molecule (GenBankAccession #U22512) encoding a portion of a human CHUK protein that is93% identical at the nucleotide level (95% identical at the amino acidlevel) with the mouse CHUK also was identified. However, neither thefunction of a CHUK protein in a cell nor a potential substrate for theputative kinase was described.

The present invention provides an isolated IκB kinase (IKK), includingisolated full length IKK catalytic subunits. For example, the inventionprovides an isolated 300 kDa or 900 kDa complex, which comprises an IKKαor an IKKβ subunit and has IκB kinase activity (see Examples I, III andIV). In addition, the invention provides is an isolated human IKKαcatalytic subunit (SEQ ID NO: 2; Example II), which contains apreviously undescribed N-terminal amino acid sequence and essentiallythe C-terminal region of human CHUK (Connelly and Marcu, supra, 1995)and phosphorylates IκBα on Ser-32 and Ser-36 and IκBβ on Ser-19 andSer-23 (DiDonato et al., supra, 1996; see, also, Regnier et al., supra,1997). The invention also provides an isolated IKKβ catalytic subunit(SEQ ID NO: 15; Example III), which shares greater than 50% amino acidsequence identity with IKKα, including conserved homology in the kinasedomain, helix-loop-helix domain and leucine zipper domain.

As used herein, the term “isolated,” when used in reference to an IκBkinase complex or to an IKK catalytic subunit of the invention, meansthat the complex or the subunit is relatively free from contaminatinglipids, proteins, nucleic acids or other cellular material normallyassociated with an IKK in a cell. An isolated 900 kDa IκB kinase complexor 300 kDa complex can be isolated, for example, by immunoprecipitationusing an antibody that binds to an IKK catalytic subunit (see ExamplesIII and IV). In addition, an isolated IKK subunit can be obtained, forexample, by expression of a recombinant nucleic acid molecule such asSEQ ID NO: 1 or SEQ ID NO: 14, or can be isolated from a cell by amethod comprising affinity chromatography using ATP or IκB as ligands(Example I) or using an anti-IKK subunit antibody. An isolated IKKcomplex or IKK subunit comprises at least 30% of the material in asample, generally about 50% or 70% or 90% of a sample, and preferablyabout 95% or 98% of a sample, as described above with respect to nucleicacids.

The amino acid sequences for MEKK1 (GenBank Accession #U48596; locusRNU48596), PKR (GenBank Accession #M35663; locus HUMP68A) and CKII(GenBank Accession #M55268 J02924; locus HUMALCKII) are different fromthe sequences of the IKK subunits disclosed herein (SEQ ID NO: 2 and SEQID NO: 15) and, therefore, are distinguishable from the presentinvention. In addition, a full length human IKKα of the invention isdistinguishable from the partial human CHUK polypeptide sequence in thatthe partial human CHUK polypeptide (Connelly and Marcu, supra, 1995;GenBank Accession #22512) lacks amino acids 1 to 31 as shown in SEQ IDNO: 2. As disclosed herein, a polypeptide having the amino acid sequenceof the partial human CHUK polypeptide does not have IκB kinase activitywhen expressed in a cell, indicating that some or all of amino acidresidues 1 to 31 are essential for kinase activity.

A full length IKK catalytic subunit of the invention is exemplified byhuman IKKα, which has an apparent molecular mass of about 85 kDa andphosphorylates IκBα on Ser-32 and Ser-36. An IKK catalytic subunit ofthe invention also is exemplified by IKKβ, which is an 87 kDapolypeptide that shares substantial amino acid sequence homology withIKKα (FIG. 3). As used herein, the term “full length,” when used inreference to an IKK subunit of the invention, means a polypeptide havingan amino acid sequence of an IKK subunit expressed normally in a cell.Such a normally expressed IKK polypeptide begins with a methionineresidue at its N-terminus (Met-1; FIG. 3), the Met-1 being encoded bythe initiator ATG (AUG) codon, and ends as a result of the terminationof translation due to the presence of a STOP codon. A full length humanIKK catalytic subunit can be a native IKK polypeptide, which is isolatedfrom a cell, or can be produced using recombinant DNA methods such as byexpressing the nucleic acid molecule shown as SEQ ID NO: 1 or SEQ ID NO:14.

The apparent molecular mass of an isolated IKK subunit can be measuredusing routine methods such as polyacrylamide gel electrophoresisperformed in the presence of sodium dodecyl sulfate (SDS-PAGE) or columnchromatography performed under reducing and denaturing conditions. Inaddition, the ability of an IKK subunit to phosphorylate IκBα on Ser-32and Ser-36 can be identified using the methods disclosed herein.

With regard to the disclosed 85 kDa and 87 kDa apparent molecular massesof human IKKα and IKKβ, it is recognized that the apparent molecularmass of a previously unknown protein as determined, for example, bySDS-PAGE is an estimate based on the relative migration of the unknownprotein as compared to the migration of several other proteins havingknown molecular masses. Thus, one investigator reasonably can estimate,for example, that an unknown protein has an apparent molecular mass of82 kDa, whereas a second investigator, looking at the same unknownprotein under substantially similar conditions, reasonably can estimatethat the protein has an apparent molecular mass of 87 kDa. Accordingly,reference herein to an IκB kinase having an apparent molecular mass of“about 85 kDa” indicates that the kinase migrates by SDS-PAGE in an 8%gel under reducing conditions in the range of 80 kDa to 90 kDa,preferably in the range of 82 kDa to 87 kDa. Furthermore, referenceherein to an 87 kDa IKKβ indicates that IKKβ has a relatively higherapparent molecular mass than the 85 kDa apparent molecular mass of IKKα.

An IKK catalytic subunit of the invention is exemplified by the isolatedfull length polypeptide comprising the amino acid sequence shown as SEQID NO: 2 or SEQ ID NO: 15. In addition, the invention provides peptideportions of an IKK subunit polypeptide, wherein such peptide portionscontain at least three contiguous amino acids as shown in SEQ ID NO: 2or SEQ ID NO: 15, and generally contain at least six contiguous aminoacids or, if desired, at least nine contiguous amino acids, as providedherein. Thus, the invention provides peptide portions of IKKα,containing, for example, at least three contiguous amino acids of SEQ IDNO: 2, including amino acid residue 30, preferably at least fourcontiguous amino acids, including amino acid residue 30, and morepreferably at least six contiguous amino acids, including amino acidresidue 30. The invention also provides a peptide portion of IKKβcomprising at least three contiguous amino acids, generally sixcontiguous amino acids, and preferably ten contiguous amino acids of SEQID NO: 15. It is recognized, however, that a peptide of the inventiondoes not consist of a polypeptide disclosed as GenBank Accession #U12473or #U22512.

A peptide portion of an IKK subunit generally is a tripeptide or larger,preferably a hexapeptide or larger, and more preferably a decapeptide orlarger, up to a contiguous amino acid sequence having a maximum lengththat lacks one or more N-terminal or C-terminal amino acids of the fulllength polypeptide (SEQ ID-NO: 2 or SEQ ID NO: 15). Thus, a peptideportion of IKKα having the amino acid sequence shown as SEQ ID NO: 2 canbe from three amino acids long to 744 amino acids long, which is oneresidue less than the full length polypeptide, except as provided above.

A peptide portion of an IKK subunit polypeptide of the invention can beproduced by any of several methods well known in the art. For example, apeptide portion of an IKK subunit can be produced by enzymatic cleavageof an IKK subunit protein, which has been isolated from a cell, using aproteolytic enzyme such as trypsin, chymotrypsin, Lys-C or the like, orcombinations of such enzymes. Such proteolytic cleavage products can beisolated using methods as disclosed in Example I, to obtain peptideportions of IKKα and IKKβ, for example. A peptide portion of an IKKsubunit also can be produced using methods of solution or solid phasepeptide synthesis or can be expressed from a nucleic acid molecule suchas a portion of the coding region of the nucleic acid sequence shown asSEQ ID NO: 1 or SEQ ID NO: 14, or can be purchased from a commercialsource.

A peptide portion of an IKK subunit can comprise the kinase domain ofthe IKK subunit and, therefore, can have the ability to phosphorylate anIκB protein. For example, a peptide portion of SEQ ID NO: 2 comprisingamino acids 15 to 301 has the characteristics of a serine-threonineprotein kinase domain (Hanks and Quinn, Meth. Enzymol. 200:38-62 (1991),which is incorporated herein by reference). Such a peptide portion of anIKK subunit can be examined for kinase activity by determining that itcan phosphorylate IκBα at Ser-32 and Ser-36 or IκBβ at Ser-19 andSer-23, using methods as disclosed herein. In addition, a peptideportion of an IKK subunit can comprise an immunogenic amino acidsequence of the polypeptide and, therefore, can be useful for elicitingproduction of an antibody that can specifically bind the IKK subunit orto an IKK complex comprising the subunit, particularly to an epitopecomprising amino acid residue 30 as shown in SEQ ID NO: 2 or to anepitope of SEQ ID NO: 15, provided said epitope is not present in a CHUKprotein. Accordingly, the invention also provides anti-IKK antibodies,which specifically bind to an epitope of an IKK complex, particularly anIKK catalytic subunit, and to IKK subunit binding fragments of suchantibodies. In addition, the invention provides cell lines producinganti-IKK antibodies or IKK-binding fragments of such antibodies.

As used herein, the term “antibody” is used in its broadest senseto-include polyclonal and monoclonal antibodies, as well as antigenbinding fragments of such antibodies. With regard to an anti-IKKantibody of the invention, the term “antigen” means an IKK catalyticsubunit protein, polypeptide or peptide portion thereof, or an IKKcomplex comprising an IKK catalytic subunit protein, polypeptide orpeptide portion thereof. Thus, it should be recognized that, while ananti-IKK antibody can bind to and, for example, immunoprecipitate an IKKcomplex, the antibody specifically binds an epitope comprising at leasta portion of an IKK catalytic subunit. An antibody of the invention alsocan be used to immunoprecipitate an IKK subunit, free of the IKKcomplex.

An anti-IKK antibody, or antigen binding fragment of such an antibody,is characterized by having specific binding activity for an epitope ofan IKK subunit of at least about 1×10⁵ M⁻¹, generally, at least about1×10⁶ M⁻¹. Thus, Fab, F(ab′)₂, Fd and Fv fragments of an anti-IKKantibody, which retain specific binding activity for an IKK subunit, areincluded within the definition of an antibody. In particular, ananti-IKK antibody can react with an epitope comprising the N-terminus ofIKKα or with an epitope of IKKβ, but not to a polypeptide having anamino acid sequence shown as residues 32 to 745 of SEQ ID NO: 2.

The term “antibody” as used herein includes naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains as described by Huse et al.,Science 246:1275-1281 (1989), which is incorporated herein by reference.These and other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known tothose skilled in the art (Winter and Harris, Immunol. Today 14:243-246(1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane,Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press,1988); Hilyard et al., Protein Engineering: A practical approach (IRLPress 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford UniversityPress 1995); each of which is incorporated herein by reference).

An anti-IKK antibody of the invention can be raised using an isolatedIKK subunit or a peptide portion thereof and can bind to a free,uncomplexed form of IKK subunit or can bind to IKK subunit when it isassociated with a 300 kDa or 900 kDa IKK complex. In addition, ananti-IKK antibody of the invention can be raised against an isolated 300kDa or 900 kDa IκB kinase complex, which can be obtained as disclosedherein. For convenience, an antibody of the invention is referred togenerally herein as an “anti-IκB kinase antibody” or an “anti-IKKantibody.” However, the skilled recognize that the various antibodies ofthe invention will have unique antigenic specificities, for example, fora free or complexed IKK subunit, or both, or for a 300 kDa or 900 kDaIκB kinase complex, or both.

Anti-IKK antibodies can be raised using as an immunogen an isolated fulllength IKK catalytic subunit, which can be prepared from natural sourcesor produced recombinantly, or a peptide portion of an IKK subunit asdefined herein, including synthetic peptides as described above. Anon-immunogenic peptide portion of an IKK catalytic subunit can be madeimmunogenic by coupling the hapten to a carrier molecule such bovineserum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressingthe peptide portion as a fusion protein. Various other carrier moleculesand methods for coupling a hapten to a carrier molecule are well knownin the art and described, for example, by Harlow and Lane, supra, 1988).It is recognized that, due to the apparently high amino acid sequenceidentity of the full length human IKKα and mouse CHUK, the amino acidsequences of IKKα polypeptides, as well as IKKβ polypeptides, likely arehighly conserved among species, particularly among mammalian species.However, antibodies to highly conserved proteins have been raisedsuccessfully, for example, in chickens. Such a method can be used toobtain an antibody to an IKK subunit, if desired.

Particularly useful antibodies of the invention include antibodies thatbind with the free, but not the complexed, form of an IKK subunit or,alternatively, with the complexed, but not free, form of an IKK subunit.Antibodies of the invention also include antibodies that bind with the300 kDa IκB kinase complex or the 900 kDa IκB kinase complex or both. Itshould be recognized, however, that an antibody specific for the 300 kDaor 900 kDa IκB kinase complex need not recognize an IKK subunit epitopein order to be encompassed within the claimed invention, since, prior tothe present disclosure, the 300 kDa and 900 kDa IKK complexes were notknown (see DiDonato et al., Nature 388:548-554 (1997)).

Antibodies of the invention that bind to an activated IKK but not to aninactive IKK, and, conversely, those that bind to an inactive form ofthe kinase but not to the activated form also are particularly useful.For example, an IKK can be activated by phosphorylation of an IKKsubunit and, therefore, an antibody that recognizes the phosphorylatedform of the IKK, but that does not bind to the unphosphorylated form canbe obtained. In addition, IKK can be activated by release of aregulatory subunit and, therefore, an antibody that recognizes a form ofthe IKK complex that is not bound to the regulatory subunit can beobtained. Such antibodies are useful for identifying the presence ofactive IKK in a cell.

An anti-IKK antibody is useful, for example, for determining thepresence or level of an IKK or of an IKK subunit in a tissue sample,which can be a lysate or a histological section. The identification ofthe presence or level of an IKK or an IKK subunit in the sample can bemade using well known immunoassay and immunohistochemical methods(Harlow and Lane, supra, 1988). An anti-IKK antibody also can be used tosubstantially purify an IκB kinase or an IKK subunit from a sample. Inaddition, an anti-IKK antibody can be used in a screening assay toidentify agents that alter the activity of an IκB kinase.

A kit incorporating an anti-IKK antibody, which can be specific for theactive or inactive form of IκB kinase or can bind to an IKK complex orto an IKK subunit, regardless of the activity state, can be particularlyuseful. Such a kit can contain, in addition to an anti-IKK antibody, areaction cocktail that provides the proper conditions for performing theassay, control samples that contain known amounts of an IKK or IKKsubunit and, if desired, a second antibody specific for the anti-IKKantibody. Such an assay also should include a simple method fordetecting the presence or amount of an IKK or an IKK subunit in a samplethat is bound to the anti-IKK antibody.

A protein such as anti-IKK antibody, as well as an IKK subunit or apeptide portion thereof, can be labeled so as to be detectable usingmethods well known in the art (Hermanson, “Bioconjugate Techniques”(Academic Press 1996), which is incorporated herein by reference; Harlowand Lane, 1988; chap. 9). For example, a protein can be labeled withvarious detectable moieties including a radiolabel, an enzyme, biotin ora fluorochrome. Reagents for labeling a protein such as an anti-IKKantibody can be included in a kit containing the protein or can bepurchased separately from a commercial source.

Following contact, for example, of a labeled antibody with a sample suchas a tissue homogenate or a histological section of a tissue,specifically bound labeled antibody can be identified by detecting theparticular moiety. Alternatively, a labeled second antibody can be usedto identify specific binding of an unlabeled anti-IKK antibody. A secondantibody generally will be specific for the particular class of thefirst antibody. For example, if an anti-IκB kinase antibody is of theIgG class, a second antibody will be an anti-IgG antibody. Such secondantibodies are readily available from commercial sources. The secondantibody can be labeled using a detectable moiety as described above.When a sample is labeled using a second antibody, the sample is firstcontacted with a first antibody, which is an anti-IKK antibody, then thesample is contacted with the labeled second antibody, which specificallybinds to the anti-IKK antibody and results in a labeled sample.

Methods for raising polyclonal antibodies, for example, in a rabbit,goat, mouse or other mammal, are well known in the art (see Example V).In addition, monoclonal antibodies can be obtained using methods thatare well known and routine in the-art (Harlow and Lane, supra, 1988).Essentially, spleen cells from a mouse immunized with an IKK complex oran IKK subunit or peptide portion thereof can be fused to an appropriatemyeloma cell line such as SP/02 myeloma cells to produce hybridomacells. Cloned hybridoma cell lines can be screened using a labeled IKKsubunit to identify clones that secrete anti-IKK monoclonal antibodies.Hybridomas expressing anti-IKK monoclonal antibodies having a desirablespecificity and affinity can be isolated and utilized as a continuoussource of the antibodies, which are useful, for example, for preparingstandardized kits as described above. Similarly, a recombinant phagethat expresses, for example, a single chain anti-IKK also provides amonoclonal antibody that can used for preparing standardized kits.

A monoclonal anti-IKK antibody can be used to prepare anti-idiotypicantibodies, which present an epitope that mimics the epitope recognizedby the monoclonal antibody used to prepare the anti-idiotypicantibodies. Where the epitope to which the monoclonal antibody includes,for example, a portion of the IKK catalytic subunit kinase domain, theanti-idiotypic antibody can act as a competitor of IκB and, therefore,can be useful for reducing the level of phosphorylation of IκB and,consequently, the activity of NF-κB.

The present invention further provides methods of identifying an agentthat can alter the association of an IKK catalytic subunit with a secondprotein, which can be an upstream activator, a downstream effector suchas IκB, an interacting regulatory protein of the IKK subunit, or aninteracting subunit associated with the 300 kDa or 900 kDa IκB kinasecomplex. As used herein, the term “associate” or “association,” whenused in reference to an IKK subunit and a second protein means that theIKK subunit and the second protein have a binding affinity for eachother such that they form a bound complex in vivo or in vitro, includingin a cell in culture or in a reaction comprising substantially purifiedreagents. For convenience, the term “bind” or “interact” is usedinterchangeably with the term “associate.”

The affinity of binding of an IKK subunit and a second protein such asan IκB or another IKK subunit or other-subunit present in an IKK complexis characterized in that it is sufficiently specific such that a boundcomplex can form in vivo in a cell or can form in vitro underappropriate conditions as disclosed herein. The formation ordissociation of a bound complex can be identified, for example, usingthe two hybrid assay or demonstrating coimmunoprecipitation of thesecond protein with the IKK subunit, as disclosed herein, or using otherwell known methods such as equilibrium dialysis. Methods fordistinguishing the specific association of an IKK subunit and a secondprotein from nonspecific binding to the IKK subunit are known in the artand, generally, include performing the appropriate control experimentsto demonstrate the absence of nonspecific protein binding.

As used herein, the term “second protein” refers to a protein thatspecifically associates with an IKK subunit (“first protein”). Such asecond protein is exemplified herein by IκB proteins, including IκBα andIκBβ, which are substrates for IκB kinase activity and are downstream ofthe IκB kinase in a signal transduction pathway that results in theregulated expression of a gene. In addition, such second proteins areexemplified by the proteins that, together with the IKK subunits, form a300 kDa or 900 kDa IκB kinase complex, which coimmunoprecipitates usingan anti-IKK antibody (see Example IV). Furthermore, since IKK subunitssuch as IKKα and IKKβ interact with each other to form homodimers orheterodimers, a-second protein also can be a second IKK subunit, whichcan be the same as or different from the “first” protein.

Agents that alter the association of an IKK catalytic subunit and asecond protein such as IκB protein or an IKK regulatory subunit can beextremely valuable, for example, for limiting excessive cytokineexpression as occurs in an acute phase response by preventing theactivation of NF-κB, thereby preventing NF-κB mediated induction ofcytokine gene expression. Where, in a drug screening assay of theinvention, the second protein is an IκB, the IKK subunit can be anyprotein involved in IκB kinase activity, including, for example, mouseCHUK (Connelly and Marcu, supra, 1995; GenBank Accession #12473), which,prior to the present disclosure, was not known to have the ability toassociate with IκB or to have IκB kinase activity.

In addition, a second protein can be a protein that is upstream of IκBkinase in a signal transduction pathway and associates with the IKKcomplex, particularly with an IKK catalytic subunit of the IKK complex.Such a second protein, which can be an upstream activator of the IκBkinase, can be identified using routine methods for identifyingprotein-protein interactions as disclosed herein. Such second proteinscan be, for example, MEKK1 or PKR or CKII, each of which has beenreported to be involved in a pathway leading to phosphorylation of IκBand activation of NF-κB, but neither of which has the characteristicsexpected of the common IκB kinase present at the point where the variousNF-κB activation pathways converge (see, for example, Lee et al., supra,1997), or can be the NF-κB-inducing kinase (NIK), which reportedly isupstream from IKK in an NF-κB activation pathway (Regnier et al., supra,1997; Malinin et al., Nature 385:540-544 (1997)).

A second protein also can be a regulatory protein, which associates withan IKK catalytic subunit in an IKK complex, either constitutively aspart of a 300 kDa or 900 kDa complex or in response to activation of apathway leading to IKK activation. Such a regulatory protein can inhibitor activate IKK activity depending, for example, on whether theregulatory protein is associated with IKK and whether the regulatoryprotein associates with an IKK catalytic subunit in a free form or aspart of an IKK complex. The regulatory protein also can be important for“docking” a catalytic IKK subunit to its substrate. The ability of aregulatory protein to associate with or dissociate from an IKK subunitor IKK complex can depend, for example, on the relative phosphorylationstate of the regulatory protein. It is recognized that an upstreamactivator of IKK also can interact with such a regulatory protein,thereby indirectly inhibiting or activating the IKK.

As disclosed herein, two copurifying proteins were isolated by ATP andIκB affinity chromatography and identified by SDS-PAGE (Example I).Partial amino acid sequences were determined and cDNA molecules encodingthe proteins were obtained (see Examples I, II and III). One of theproteins has an apparent molecular mass of 85 kDa. Expression in a cellof a cDNA molecule encoding the 85 kDa protein resulted in increasedNF-κB activity following cytokine induction as compared to controlcells, whereas expression of the antisense of this cDNA decreased thebasal NF-κB activity in the cells and prevented cytokine induction ofNF-κB activity. Immunoprecipitation of the 85 kDa protein resulted inisolation of the IKK complex, the kinase activity of which wasstimulated rapidly in response to TNF or to IL-1. Based on thesefunctional analyses, the 85 kDa protein was determined to be a componentof the 900 kDa IκB kinase complex and has been designated IKKα (SEQ IDNO: 2). The second protein, which copurified with the 85 kDa IκB kinase,has an apparent molecular mass of 87 kDa and shares greater than 50%amino acid sequence identity with IKKα and has been designated IKKβ (SEQID NO: 15).

The ability of the 85 kDa and 87 kDa IKK subunits to associate withother proteins such as a regulatory subunit as well as with IκB issuggested, for example, by the presence in the IκB kinase of twodifferent protein binding domains, a helix-loop-helix domain and aleucine zipper domain (see Connelly and Marcu, supra, 1995; see, also,FIG. 3). While the leucine zipper motif mediates homotypic andheterotypic interactions between IKKα and IKKβ, the helix-loop-helixmotif serves as a binding site for regulatory proteins necessary for IκBkinase activation.

A screening assay of the invention provides a means to identify an agentthat alters the association of an IKK complex or an IKK catalyticsubunit with a second protein such as the regulatory subunits discussedabove. As used herein, the term “modulate” or “alter” when used inreference to the association of an IKK and a second protein, means thatthe affinity of the association is increased or decreased with respectto a steady state, control level of association, i.e., in the absence ofan agent. Agents that can alter the association of an IKK with a secondprotein can be useful for modulating the level of phosphorylation of IκBin a cell, which, in turn, modulates the activity of NF-κB in the celland the expression of a gene regulated by NF-κB. Such an agent can be,for example, an anti-idiotypic antibody as described above, which caninhibit the association of an IKK and IκB. A peptide portion of IκBαcomprising amino acids 32 to 36, but containing substitutions for Ser-32and Ser-36, is another example of such an agent, since the peptide cancompete with IκBα binding to IKK, as is the corresponding peptide ofIκBβ.

A screening assay of the invention also is useful for identifying agentsthat directly alter the activity of an IKK. While such an agent can act,for example, by altering the association of an IKK complex or IKKcatalytic subunit with a second protein, the agent also can act directlyas a specific activator or inhibitor of IKK activity. Specific proteinkinase inhibitors include, for example, staurosporin, the heat stableinhibitor of cAMP-dependent protein kinase, and the MLCK inhibitor,which are known in the art and commercially available. A library ofmolecules based, generally, on such inhibitors or on ATP or adenosinecan be screened using an assay of the invention to obtain agents thatdesirably modulate the activity of an IKK complex or an IKK subunit.

As disclosed herein, IKK activity can be measured by identifyingphosphorylation, for example, of IκBα, either directly or using anantibody specific for the Ser-32 and Ser-36 phosphorylated form of IκBα.An antibody that binds to IκBα that is phosphorylated on Ser-32, forexample, can be purchased from a commercial source (New England Biolabs;Beverly Mass.). Cultured cells can be exposed to various agentssuspected of having the ability to directly alter IKK activity, thenaliquots of the cells either are collected or are treated with aproinflammatory stimulus such as a cytokine, and collected. Thecollected cells are lysed and the kinase is immunoprecipitated using ananti-IKK antibody. A substrate such as IκBα or IκBβ is added to theimmunocomplex and the ability of the IKK to phosphorylate the substrateis determined as described above. If desired, the anti-IKK antibodyfirst can be coated onto a plastic surface such as in 96 well plates,then the cell lysate is added to the wells under conditions that allowbinding of IKK by the antibody. Following washing of the wells, IKKactivity is measured as described above. Such a method is extremelyrapid and provides the additional advantage that it can be automated forhigh through-put assays.

A screening assay of the invention is particularly useful to identify,from among a diverse population of molecules, those agents that modulatethe association of an IKK complex or an IKK catalytic subunit andanother protein (referred to herein as a “second protein”) or thatdirectly alter the activity of IKK. Methods for producing librariescontaining diverse populations of molecules, including chemical orbiological molecules such as simple or complex organic molecules,peptides, proteins, peptidomimetics, glycoproteins, lipoproteins,polynucleotides, and the like, are well known in the art (Huse, U.S.Pat. No. 5,264,563, issued Nov. 23, 1993; Blondelle et al., Trends Anal.Chem. 14:83-92 (1995); York et al., Science 274:1520-1522 (1996); Goldet al., Proc. Natl. Acad. Sci., USA 94:59-64 (1997); Gold, U.S. Pat. No.5,270,163, issued Dec. 14, 1993). Such libraries also can be obtainedfrom commercial sources.

Since libraries of diverse molecules can contain as many as 10¹⁴ to 10¹⁵different molecules, a screening assay of the invention provides asimple means for identifying those agents in the library that canmodulate the association of an IKK and a second protein or can alter theactivity of an IKK. In particular, a screening assay of the inventioncan be automated, which allows for high through-put screening ofrandomly designed libraries of agents to identify those particularagents that can modulate the ability of an IKK and a second protein toassociate or that alter the activity of the IKK.

A drug screening assay of the invention utilizes an IKK complex, whichcan be isolated as disclosed herein; or an IKK subunit, which can beexpressed, for example, from a nucleic acid molecule encoding the aminoacid sequence shown in SEQ ID NO: 2 or in SEQ ID NO: 15; or can bepurified as disclosed herein; or can utilize an IKK subunit fusionprotein such as an IKKα-glutathione-S-transferase (GST) orIKKβ-histidine₆ (HIS6) fusion protein, wherein the GST or HIS6 is linkedto the IKK subunit and comprises a tag (see Example VI). The IKK or IKKsubunit fusion protein is characterized, in part, by having an affinityfor a solid substrate as well as having the ability to specificallyassociate with an appropriate second protein such as an IκB protein. Forexample, when an IKK catalytic subunit is used in a screening assay, thesolid substrate can contain a covalently attached anti-IKK antibody,provided that the antibody binds the IKK subunit without interferingwith the ability of the IKK subunit to associate with the secondprotein. Where an IKKα-GST fusion protein, for example, is used in sucha screening assay, the solid substrate can contain covalently attachedglutathione, which is bound by the GST tag component of the fusionprotein. If desired, the IKK subunit or IKK subunit fusion protein canbe part of an IKK complex in a drug screening assay of the invention.

A drug screening assay to identify an agent that alters the associationof an IKK complex or an IKK subunit and a second protein can beperformed by allowing, for example, the IKK complex or IKK subunit,which can be a fusion protein, to bind to the solid support, then addingthe second protein, which can be an IκB such as IκBα, and an agent to betested, under conditions suitable for the association of the IKK andIκBα in the absence of a drug (see Example VI). As appropriate, the IKKcan be activated or inactivated as disclosed herein and, typically, theIKK or the second protein is detectably labeled so as to facilitateidentification of the association. Control reactions, which contain orlack either, the IKK component, or the IκB protein, or the agent, orwhich substitute the IκB protein with a second protein that is known notto associate specifically with the IKK, also are performed. Followingincubation of the reaction mixture, the amount of IκBα specificallybound to the IKK in the presence of an agent can be determined andcompared to the amount of binding in the absence of the agent so thatagents that modulate the association can be identified.

An IKK subunit such as IKKα or IKKβ used in a screening assay can bedetectably labeled with a radionuclide, a fluorescent label, an enzyme,a peptide epitope or other such moiety, which facilitates adetermination of the amount of association in a reaction. By comparingthe amount of specific binding of an IKK subunit or an IKK complex andIκB in the presence of an agent as compared to the control level ofbinding, an agent that increases or decreases the binding of the IKK andthe IκB can be identified. In comparison, where a drug screening assayis used to identify an agent that alters the activity of an IKK, thedetectable label can be, for example, γ-³²P-ATP, and the amount of³²P-IκB can be detected as a measure of IKK activity. Thus, the drugscreening assay provides a rapid and simple method for selecting agentsthat desirably alter the association of an IKK and a second protein suchas an IκB or for altering the activity of an IKK. Such agents can beuseful, for example, for modulating the activity of NF-κB in a cell and,therefore, can be useful as medicaments for the treatment of a pathologydue, at least in part, to aberrant NF-κB activity.

The method for performing a drug screening assay as disclosed hereinalso provides a research tool for identifying a target of a drug that isor can be used therapeutically to ameliorate an undesirable inflammatoryor immune response, but for which the target of the drug is not known.Cytokine restraining agents, for example, are a class of agents that canalter the level of cytokine expression (U.S. Pat. No. 5,420,109, issuedMay 30, 1995) and can be used to treat various pathologies, includingpatho-immunogenic diseases such as rheumatoid arthritis and thoseinduced by exposure to bacterial endotoxin such as occur in septic shock(see, also, WO96/27386, published Sep. 12, 1996).

The specific cellular target upon which a cytokine restraining agentacts has not been reported. However, the myriad of pathologic effectsameliorated by such agents are similar to various pathologies associatedwith aberrant NF-κB activity, suggesting that cytokine restrainingagents may target an effector molecule in a NF-κB signal transductionpathway. Thus, one potential target of a cytokine restraining agent canbe an IκB kinase, particularly an IKK catalytic subunit of the kinase.Accordingly, a screening assay of the invention can be used to determinewhether a cytokine restraining agent alters the activity of IκB kinaseor alters the association of an IKK and a second protein such as IκB. Ifit is determined that a cytokine restraining agent has such an effect,the screening assay then can be used to screen a library of cytokineregulatory agents to identify those having desirable characteristics,such as those having the highest affinity for the IKK.

The invention also provides a method of obtaining an isolated IKKcomplex or an IKK catalytic subunit. For example, a 300 kDa or a 900 kDaIKK complex, comprising an IKKα subunit can be isolated from a sample byimmunoprecipitation using an anti-IKKα antibody or by tagging the IKKαand using an antibody specific for the tag (see Examples III and IV). Inaddition, an IKK catalytic subunit can be isolated from a sample by 1)incubating the sample containing the IKK subunit with ATP, which isimmobilized on a matrix, under conditions suitable for binding of theIKK subunit to the ATP; 2) obtaining from the immobilized ATP a fractionof the sample containing the IKK subunit; 3) incubating the fractioncontaining the IKK subunit with an IκB, which is immobilized on amatrix, under conditions suitable for binding of the IKK subunit to theIκB; and 4) obtaining from the immobilized IκB an isolated IKK catalyticsubunit. Such a method of isolating an IKK subunit is exemplified hereinby the use of ATP affinity chromatography and IκBα affinitychromatography to isolate IKKα or IKKβ from a sample of HeLa cells (seeExample I).

The skilled artisan will recognize that a ligand such as ATP or an IκBor an anti-IKK antibody also can be immobilized on various othermatrices, including, for example, on magnetic beads, which provide arapid and simple method of obtaining a fraction containing an ATP- or anIκB-bound IKK complex or IKK subunit or an anti-IκB kinase-bound IKKfrom the remainder of the sample. Methods for immobilizing a ligand suchas ATP or an IκB or an antibody are well known in the art (Haystead etal., Eur. J. Biochem. 214:459-467 (1993), which is incorporated hereinby reference; see, also, Hermanson, supra, 1996). Similarly, the artisanwill recognize that a sample containing an IKK complex or an IKK subunitcan be a cell, tissue or organ sample, which is obtained from an animal,including a mammal such as a human, and prepared as a lysate; or can bea bacterial, insect, yeast or mammalian cell lysate, in which an IKKcatalytic subunit is expressed from a recombinant nucleic acid molecule.As disclosed herein, a recombinantly expressed IKKα or IKKβ such as atagged IKKα or IKKβ associates into an active 300 kDa and 900 kDa IKKcomplex (see Examples III and IV).

The invention also provides a method of identifying a second proteinthat associates with an IKK complex, particularly with an IKK subunit. Atranscription activation assay such as the yeast two hybrid system isparticularly useful for the identification of protein-proteininteractions (Fields and Song, Nature 340:245-246 (1989), which isincorporated herein by reference). In addition, the two hybrid assay isuseful for the manipulation of protein-protein interaction and,therefore, also is useful in a screening assay to identify agents thatmodulate the specific interaction.

A transcription activation assay such as the two hybrid assay also canbe performed in mammalian cells (Fearon et al., Proc. Natl. Acad. Sci.,USA 89:7958-7962 (1992), which is incorporated herein by reference).However, the yeast two hybrid system provides a particularly usefulassay due to the ease of working with yeast and the speed with which theassay can be performed. Thus, the invention also provides methods ofidentifying proteins that can interact with an IKK subunit, includingproteins that can act as upstream activators or downstream effectors ofIKK activity in a signal transduction pathway mediated by the IKK orproteins that bind to and regulate the activity of the IKK. Suchproteins that interact with an IKK catalytic subunit can be involved,for example, in tissue specific regulation of NF-κB activation orconstitutive NF-κB activation and consequent gene expression.

The conceptual basis for a transcription activation assay is predicatedon the modular nature of transcription factors, which consist offunctionally separable DNA-binding and trans-activation domains. Whenexpressed as separate proteins, these two domains fail to mediate genetranscription. However, the ability to activate transcription can berestored if the DNA-binding domain and the trans-activation domain arebridged together through a protein-protein interaction. These domainscan be bridged, for example, by expressing the DNA-binding domain andtrans-activation domain as fusion proteins (hybrids), where the proteinsthat are appended to these domains can interact with each other. Theprotein-protein interaction of the hybrids can bring the DNA-binding andtrans-activation domains together to create a transcriptionallycompetent complex.

One adaptation of the transcription activation assay, the yeast twohybrid system, uses S. cerevisiae as a host cell for vectors thatexpress the hybrid proteins. For example, a yeast host cell containing areporter lacZ gene linked to a LexA operator sequence can be used toidentify specific interactions between an IKK subunit and a secondprotein, where the DNA-binding domain is the LexA binding domain, whichbinds the LexA promoter, and the trans-activation domain is the B42acidic region. When the LexA domain is bridged to the B42transactivation domain through the interaction of the IKK subunit with asecond protein, which can be expressed, for example, from a cDNAlibrary, transcription of the reporter lacZ gene is activated. In thisway, proteins that interact with the IKK subunit can be identified andtheir role in a signal transduction pathway mediated by the IKK can beelucidated. Such second proteins can include additional subunitscomprising the 300 kDa or 900 kDa IKK complex.

In addition to identifying proteins that were not previously known tointeract with an IKK, particularly with an IKKα or IKKβ subunit, atranscription activation assay such as the yeast two hybrid system alsois useful as a screening assay to identify agents that alter associationof an IKK subunit and a second protein known to bind the IKK. Thus, asdescribed above for in vitro screening assays, a transcriptionactivation assay can be used to screen a panel of agents to identifythose agents particularly useful for altering the association of an IKKsubunit and a second protein in a cell. Such agents can be identified bydetecting an altered level of transcription of a reporter gene, asdescribed above, as compared to the level of transcription in theabsence of the agent. For example, an agent that increases theinteraction between an IKK subunit and IκB can be identified by anincreased level of transcription of the reporter gene as compared to thecontrol level of transcription in the absence of the agent. Such amethod is particularly useful because it identifies an agent that altersthe association of an IKK subunit and a second protein in a living cell.

In some cases, an agent may not be able to cross the yeast cell walland, therefore, cannot enter the yeast cell to alter a protein-proteininteraction. The use of yeast spheroplasts, which are yeast cells thatlack a cell wall, can circumvent this problem (Smith and Corcoran, InCurrent Protocols in Molecular Biology (ed. Ausubel et al.; Green Publ.,NY 1989), which is incorporated herein by reference). In addition, anagent, upon entering a cell, may require “activation” by a cellularmechanism that may not be present in yeast. Activation of an agent caninclude, for example, metabolic processing of the agent or amodification such as phosphorylation of the agent, which can benecessary to confer activity upon the agent. In this case, a mammaliancell line can be used to screen a panel of agents (Fearon et al., supra,1992).

An agent that alters the catalytic activity of an IKK or that alters theassociation of an IKK subunit or IKK complex and a second protein suchas an IκB or an IKK regulatory subunit or an upstream activator of anIKK can be useful as a drug to reduce the severity of a pathologycharacterized by aberrant NF-κB activity. For example, a drug thatincreases the activity of an IKK or that increases the affinity of anIKK catalytic subunit and IκBα can increase the amount of IκBαphosphorylated on Ser-32 or Ser-36 and, therefore, increase the amountof active NF-κB and the expression of a gene regulated by NF-κB, sincethe drug will increase the level of phosphorylated IκBα in the cell,thereby allowing NF-κB translocation to the nucleus. In contrast, a drugthat decreases or inhibits the catalytic activity of an IKK or theassociation of an IKK catalytic subunit and IκBα can be useful where itis desirable to decrease the level of active NF-κB in a cell and theexpression of a gene induced by activated NF-κB. It should be recognizedthat an antisense IKK subunit molecule of the invention also can be usedto decrease IKK activity in a cell by reducing or inhibiting expressionof the IKK subunit or by reducing or inhibiting its responsiveness to aninducing agent such as TNFα, Il-1 or phorbol ester (see Example II).Accordingly, the invention also provides methods of treating anindividual suffering from a pathology characterized by aberrant NF-κBactivity by administering to the individual an agent that modulates thecatalytic activity of an IKK or that alters the association of an IKKsubunit and a second protein such as IκB or a subunit of a 300 kDa or900 kDa IKK complex that interacts with the IKK subunit.

An agent that decreases the activity of an IKK or otherwise decreasesthe amount of IκB phosphorylation in a cell can reduce or inhibit NF-κBmediated gene expression, including, for example, the expression ofproinflammatory molecules such as cytokines and other biologicaleffectors involved in an inflammatory, immune or acute phase response.The ability to reduce or inhibit such gene expression can beparticularly valuable for treating various pathological conditions suchas rheumatoid arthritis, asthma and septic shock, which arecharacterized or exacerbated by the expression of such proinflammatorymolecules.

Glucocorticoids are potent anti-inflammatory and immunosuppressiveagents that are used clinically to treat various pathologic conditions,including autoimmune diseases such as rheumatoid arthritis, systemiclupus erythematosis and asthma. Glucocorticoids suppress the immune andinflammatory responses, at least in part, by increasing the rate of IκBαsynthesis, resulting in increased cellular levels of IκBα, which bind toand inactivate NF-κB (Scheinman et al., Science 270:283-286 (1995);Auphan et al., Science 270:286-290 (1995)). Thus, glucocorticoidssuppress NF-κB mediated expression of genes encoding, for example,cytokines, thereby suppressing the immune, inflammatory and acute phaseresponses. However, glucocorticoids and glucocorticoid-like steroidsalso are produced physiologically and are required for normal growth anddevelopment. Unfortunately, prolonged treatment of an individual withhigher than physiological amounts of glucocorticoids produces clinicallyundesirable side effects. Thus, the use of an agent that alters theactivity of an IKK or that alters the association of an IKK complex orIKK subunit and a second protein, as identified using a method of theinvention, can provide a means for selectively altering NF-κB activitywithout producing some of the undesirable side effects associated withglucocorticoid treatment.

Inappropriate regulation of Rel/NF-κB transcription factors isassociated with various human diseases. For example, many viruses,including human immunodeficiency virus-1 (HIV-1), herpes simplex virus-1(HSV-1) and cytomegalovirus (CMV) contain genes regulated by a κBregulatory element and these viruses, upon infecting a cell, utilizecellular Rel/NF-κB transcription factors to mediate viral geneexpression (Siebenlist et al., supra, 1994). Tat-mediated transcriptionfrom the HIV-1 enhancer, for example, is decreased if the NF-κB and SP1binding sites are deleted from the enhancer/promotor region, indicatingthat Tat interacts with NF-κB, SP1 or other transcription factors boundat this site to stimulate transcription (Roulston et al., Microbiol.Rev. 59:481-505 (1995)). In addition, chronic HIV-1 infection, andprogression to AIDS, is associated with the development of constitutiveNF-κB DNA binding activity in myeloid cells (Roulston et al., supra,1995). Thus, a positive autoregulatory loop is formed, whereby HIV-1infection results in constitutively active NF-κB, which inducesexpression of HIV-1 genes (Baeuerle and Baltimore, Cell 87:13-20 (1996).Constitutive NF-κB activation also may protect cells against apoptosis,preventing clearance of virus-infected cells by the immune system (Liuet al., supra, 1996).

An agent that decreases the activity of an IKK or that alters theassociation of an IKK and a second protein such that IκB phosphorylationis decreased can be useful for reducing the severity of a viralinfection such as HIV-1 infection in an individual by providingincreased levels of unphosphorylated IκB in virus-infected cells. Theunphosphorylated IκB then can bind to NF-κB in the cell, therebypreventing nuclear translocation of the NF-κB and viral gene expression.In this way, the rate of expansion of the virus population can belimited, thereby providing a therapeutic advantage to the individual.

In addition, the decreased level of NF-κB activity may allow thevirus-infected cell to undergo apoptosis, resulting in a decrease in theviral load in the individual. As such, it can be particularly useful totreat virus-infected cells ex vivo with an agent identified using amethod of the invention. For example, peripheral blood mononuclear cells(PBMCs) can be collected from an HIV-1 infected individual and treatedin culture with an agent that decreases the activity of an IKK or altersthe association of an IKK complex or an IKK catalytic subunit with anIκB. Such a treatment can be useful to purge the PBMCs of thevirus-infected cells by allowing apoptosis to proceed. The purgedpopulation of PBMCs then can be expanded, if desired, and readministeredto the individual.

Rel/NF-κB proteins also are involved in a number of different types ofcancer. For example, the adhesion of cancer cells to endothelial cellsis increased due to treatment of the cancer cells with IL-1, suggestingthat NF-κB induced the expression of cell adhesion molecules, whichmediated adherence of the tumor cells to the endothelial cells; agentssuch as aspirin, which decrease NF-κB activity, blocked the adhesion byinhibiting expression of the cell adhesion molecules (Tozawa et al.,Cancer Res. 55:4162-4167 (1995)). These results indicate that an agentthat decreases the activity of an IKK or that decrease the associationof an IKK and IκB or of an IKK subunit and a second protein, forexample, a second protein present in an IKK complex, can be useful forreducing the likelihood of metastasis of a tumor in an individual.

As discussed above for virus-infected cells, constitutive NF-κBactivation also may protect tumor cells against programmed cell death aswell as apoptosis induced by chemotherapeutic agents (Liu et al., supra,1996; Baeuerle and Baltimore, Cell 87:13-20 (1996)). Thus, an agent thatdecreases IKK activity or that decreases the association of IKK and IκBalso can be useful for allowing programmed cell death to occur in atumor cell by increasing the level of unphosphorylated IκB, which canbind NF-κB and decrease the level of active NF-κB in the tumor cell.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Identification and Characterization of a Human IκB KinaseComplex and IKK Subunits

This example provides a method for identifying and isolating a cytokineresponsive protein kinase complex that phosphorylates IκB, whichregulates NF-κB activity, and catalytic subunits of the protein kinasecomplex.

A. Kinase Assays:

Kinase assays were performed using GST fusion proteins containing aminoacid residues 1 to 54 of IκB. The fusion proteins were linked toglutathione SEPHAROSE and the beads were used directly in the assays. Atearlier stages in the purification of the IKK activity, the beads werewashed prior to loading onto the gel to minimize contributions fromother proteins. In some of the later characterization of highly purifiedmaterial, soluble fusion protein was used.

Three distinct substrates for the IKK activity were used: 1) substrate“WT” contained amino acid residues 1 to 54 of IκBα; 2) substrate “AA”contained amino acid residues 1 to 54 of IκBα, except that Ser-32 (S32)and S36 were replaced with Ala-32 (A32) and A36, respectively; and 3)substrate “TT” contained amino acid residues 1 to 54 of IκBα, exceptthat S32 and S36 were replaced with Thr-32 (T32) and T36, respectively(DiDonato et al., Mol. Cell. Biol. 16:1295-1304 (1996)). Each substratewas expressed as a GST fusion protein. The physiologic, inducible IκBkinase is specific for S32 and S36 (WT) in IκBα, but does not recognizethe TT or AA mutants (DiDonato et al., Mol. Cell. Biol. 16:1295-1304(1996)).

Kinase assays were carried out in 20 mM HEPES (pH 7.5-7.6), 20 mMβglycerophosphate (β-GP), 10 mM MgCl₂, 10 mM PNPP, 100 μM Na₃VO₄, 2 mMdithiothreitol (DTT), 20 μM ATP, 10 μg/ml aprotinin. NaCl concentrationwas 150-200 mM and the assays were carried out at 30° C. for 30 min.Fractionation was performed by SDS-PAGE, followed by quantitation byphosphoimager analysis.

B. Purification of IKK Complex and IKK Subunits:

The protein purification buffer (Buffer A) consisted of 20 mM Tris (pH7.6, measured at RT), 20 mM NaF, 20 mM β-GP, 1 mM PNPP, 500 μM Na₃VO₄, 2mM DTT, 2.5 mM metabisulfite, 5 mM benzamidine, 1 mM EDTA, 0.5 mM EGTA,1 mM PMSF, and 10% glycerol. Brij-35 was added as indicated. Cell lysisbuffer was Buffer A containing an additional 19 mM PNPP, 20 mM β-GP and500 μM Na₃VO₄, and 20 μg/ml aprotinin, 2.5 μg/ml leupeptin, 8.3 μg/mlbestatin, 1.7 μg/ml pepstatin.

Purification was performed using 5 to 130 liters of HeLa S3 cells. Forillustration, the procedure for a 15 liter preparation is presented. Allpurification steps were performed in a cold room at 4° C.

In order to activate the IKK, cells were stimulated with TNFα prior topurification. TNFα was either recombinant TNFα, which was purchased fromR&D Systems and used at 20 ng/ml, or HIS6-tagged TNFα, which wasexpressed and partially purified from E. coli and used at 5 μg/ml.TNFα-induced HeLa S3 cell killing activity assays were performed in thepresence of cycloheximide and indicated that the partially purifiedHIS6-tagged TNFα had approximately one-tenth the activity of thecommercial TNFα.

Fifteen liters of HeLa S3 cells were grown in suspension in high glucoseDulbecco's modified Eagle's medium supplemented with 10% calf serum, 2mg/ml L-glutamine, 100 U/ml penicillin/streptomycin, 0.11 mg/ml sodiumpyruvate, and 1× nonessential amino acids (Irvine Scientific; IrvineCalif.). Cell density was approximately 5×10⁵ cells/ml at the time ofcollection. Cells were concentrated 10-fold by centrifugation.stimulated for 5 min with TNFα at 37° C., then diluted with 2.5 volumesof ice cold phosphate buffered saline (PBS) containing 50 mM NaF andpelletted at 2000×g. The cell pellet was washed once with ice coldPBS/50 mM NaF, then suspended in lysis buffer, quick frozen in liquidnitrogen and stored at ⁻80° C.

For purification of IκB kinase, cells were thawed and cytoplasmicextract prepared. Lysis was achieved by 40 strokes in an all glassDounce homogenizer (pestle A) in lysis buffer containing 0.05% NP-40 onice. The homogenate was centrifuged at 12,000 rpm for 19 min in aBeckman SS34 rotor at 4° C.

Supernatant was collected and centrifuged at 38,000 rpm for 80 min in aBeckman 50.1 Ti rotor at 4° C. The supernatant (S100 fraction) was quickfrozen in liquid nitrogen and stored at −80° C. Small aliquots of S100material, prepared from either unstimulated HeLa cells or from TNFαstimulated cells, were purified in a single passage over a SUPEROSE 6gel filtration column (1.0×30 cm; Pharmacia; Uppsalla Sweden)equilibrated in Buffer A containing 0.1% Brij-35 and 300 mM NaCl andeluted at a flow rate of 0.3 ml/min. 0.6 ml fractions were collected andkinase assays were performed on an aliquot of each fraction. The highmolecular weight material (fractions 16-20) contained TNFα-inducible IKKactivity, which is specific for the WT substrate.

110 ml of S100 material (900 mg of protein; Bio-Rad Protein Assay) waspumped onto a Q-SEPHAROSE FAST FLOW column (56 ml bed volume, 2.6 cm ID)equilibrated at 2 ml/min with Buffer A containing 0.1% Brij-35. Afterthe sample was loaded, the column was washed with 100 ml of Buffer Acontaining 0.1% Brij-35 and 100 mM NaCl, then a linear NaCl gradient wasrun from 100-300 mM. The gradient volume was 500 ml and the flow ratewas 2 ml/min. Ten ml fractions were collected and the kinase assay wasperformed on those fractions that eluted during the gradient. Fractionscorresponding to the TNFα-inducible IKK activity (fractions 30-42; i.e.,20-32 of the gradient portion) were pooled. The pooled materialcontained 40 mg of protein.

The pooled material was diluted to 390 ml by addition of Buffer Acontaining 0.1% Brij-35 and loaded onto a pre-equilibrated 5 ml HITRAP Qcolumn (Pharmacia) at a flow rate of 4 ml/min. Following sample loading,the column was washed with 20 ml of Buffer A containing 0.1% Brij-35.The protein was eluted at 1 ml/min isocratically in Buffer A containing0.1% Brij-35 and 300 mM NaCl and 1 ml fractions were collected.Protein-containing fractions were identified using the BioRad assay andwere collected and pooled to yield 4 ml of solution. Previouslyperformed control experiments demonstrated that the IKK activitydirectly correlated with protein concentration.

The pooled material was diluted 1:1 with ATP column buffer (20 mM HEPES(pH 7.3), 50 mM β-GP, 60 mM MgCl₂, 1 mM Na₂VO₄, 1.5 mM EGTA, 1 mM DTT,10 μg/ml aprotinin), then passed 4 times over a γ-ATP affinity columnhaving 4 ml bed volume (Haystead et al., supra, 1993); the column hadbeen prewashed with 2 M NaCl, 0.25% Brij-35 and equilibrated with 10 bedvolumes of ATP column buffer containing 0.05% Brij-35 at a flow rate of0.5 ml/min. Following loading of the sample, the column was washed with10 ml of ATP column buffer containing 0.05% Brij-35, then with 10 ml ATPcolumn buffer containing 0.05% Brij-35 and 250 mM NaCl.

Bound material was eluted in 10 ml of ATP column buffer containing 0.05t Brij-35, 250 mM NaCl and 10 mM ATP (elution buffer). Elution wasperformed by passing 5 ml of elution buffer through the column, allowingthe column to incubate, capped, for 20 min, then passing an additional 5ml of elution buffer through the column. The samples were pooled toyield 10 ml.

The 10 ml pooled sample from the ATP column was diluted with 30 mlBuffer A containing 0.1% Brij-35 and loaded onto a 1 ml HITRAP Q column(Pharmacia) at 1 ml/min. The column was eluted at 0.4 ml/min with BufferA containing 0.1% Brij-35 and 300 mM NaCl. 0.2 ml fractions werecollected and the four protein-containing fractions were pooled (0.5mg). The pooled material was concentrated to 200 μl on a 10K NANOSEPconcentrator (Pall/Filtron) and loaded onto a SUPEROSE 6 gel filtrationcolumn (1.0×30 cm). The SUPEROSE 6 column was equilibrated in Buffer Acontaining 0.1% Brij-35 and 300 mM NaCl and run at a flow rate of 0.3ml/min; 0.6 ml fractions were collected. Fractions 17, 18 and 19contained kinase activity.

Based on silver stained SDS-PAGE gels, the final purified materialconsisted of approximately 20 μg to 40 μg of total protein, of whichapproximately 2 μg corresponded to the 85 kDa band, later designatedIKKα (see Example II). A second band migrating at 87 kDa was laterdesignated IKKβ (see Example III). The total time from the thawing ofthe S100 material until the collection of fractions from the gelfiltration column was 24 hours.

C. Confirmation of IKK Purification:

Since the 85 kDa IKKα band identified by the kinase assay following theabove procedure contained only about 10% of the total purified protein,three additional criteria were used to confirm that the identified bandwas an intrinsic component of the IKK complex.

In one procedure, the elution profile of the SUPEROSE 6 column wasanalyzed by silver stained 8% SDS-PAGE gels, then compared to the kinaseactivity profile. For this analysis, 0.3 ml fractions were collectedfrom the SUPEROSE 6 column, then separated by 8% SDS-PAGE and silverstained. This comparison confirmed that a single band of 85 kDacorrelated precisely with the elution of IKK activity.

In a second procedure, the IKK activity was further purified on asubstrate affinity column at 4° C. A GST fusion protein was preparedcontaining the A32/A36 1 to 54 amino acid sequence of IκBα repeated 8times (GST-(8X-AA)). The GST-(8X-AA) then was covalently linked to aCNBr activated SEPHAROSE 4B resin to produce the substrate affinityresin.

IKK-containing material was diluted into Buffer A to yield a finalconcentration of 70 mM NaCl, 0.025% Brij-35, then added to the substrateaffinity resin at a ratio of 4:1 (solution:swollen beads). The resin wassuspended and the mixture rotated gently overnight in a small column at4° C. The resin was allowed to settle for 30 min, then the column waseluted by gravity. The column was washed with 4 bed volumes Buffer Acontaining 0.02% Brij-35, then the resin was suspended with 1.1 bedvolumes of Buffer A containing 600 mM NaCl and 0.1% Brij-35. The resinwas allowed to settle for 40 min, then gravity elution was performed.The column was washed with an additional 1.1 bed volumes of Buffer Acontaining 600 mM NaCl and 0.1% Brij-35 and the two fractions werepooled.

The IκBα substrate affinity column was used for two separateexperiments. In one experiment, the material that eluted from the finalSUPEROSE 6 column was further purified on the IκBα substrate affinitycolumn. In the second experiment, material obtained after the initialQ-SEPHAROSE column was purified on the IκBα substrate affinity column.The Q-SEPHAROSE bound fraction then was further purified on the ATPcolumn and the SUPEROSE 6 column (see above).

Analysis of the purified material from these two experiments by silverstained SDS-PAGE gels revealed different protein profiles. However,comparison of these profiles revealed only two bands common to bothpreparations, one of which was confirmed to be the same 85 kDa IKKα bandthat was identified by the SUPEROSE 6 profile analysis andcofractionated with IκB kinase activity. The other band, which was 87kDa in size, later was identified as IKK. In several differentexperiments, the 85 kDa protein and 87 kDa protein were specificallypurified by the substrate affinity column in what appeared to be anequimolar ratio.

In a third procedure, purified IKK was treated with excess phosphatase,which inactivates the IKK, then reactivated by addition of asemi-purified HeLa extract. Phosphatase inactivation was performed byadding excess protein phosphatase 2A catalytic domain (PP2A) to purifiedIκB kinase in 50 mM Tris (pH 7.6), 50 mM NaCl, 1 mM MgCl₂, thenequilibrating the reaction for 60 min at 30° C. 1.25 μM okadaic acid wasadded to completely inactivate the phosphatase and the phosphataseinactivated material was used in standard kinase assays and to performthe reactivation and phosphorylation procedure.

Cytoplasmic extract was prepared using HeLa S3 cells. The cells werestimulated with TNFα for 5 min, then harvested in lysis buffercontaining 0.1% NP-40 and 0.15 M NaCl. Reactivation was performed at 30°C. in kinase buffer for 60 min in the absence of (γ-³²P)ATP. Samplescontaining only cold ATP were used for kinase activity assays.Reactivation by the HeLa cell extract was performed in the presence of(γ-³²P)ATP, then the sample was separated by 8% SDS-PAGE and examined byautoradiography. A band of approximately 86 kDa was phosphorylated inthe reactivated material and, associated with the reactivationprocedure, was restoration of the IKK activity.

D. Partial Amino Acid Sequences of IKKα and IKKβ

Following SDS-PAGE as described above, the 85 kDa IKKα and 87 kDa IKKβbands were excised from the gel and submitted for internal peptidesequencing analysis. From the IKKα polypeptide, the sequences of twoproteolytic fragments were identified, as follows: KIIDLLPK (SEQ ID NO:3) and KHR(D/A)LKPENIVLQDVG(P/G)K (SEQ ID NO: 4). Where a residue couldnot be unambiguously determined, an “X” was used to indicate no aminoacid could be determined and parentheses were used to delimit aminoacids that could not be distinguished. Since Lys-C protease was used todigest the protein, the presence of lysine residues at the N-termini ofthe peptides was inferred. From the 87 kDa IKKβ band, the sequences offive proteolytic fragments were determined (see FIG. 3, underlined; see,also, Example III).

EXAMPLE II Identification and Characterization of a Full Length HumanIKKα Subunit

This example provides methods for isolating a nucleic acid moleculeencoding the IKKα subunit and for characterizing the functional activityof the subunit.

A. Cloning of cDNA Encoding Human IKKα:

Degenerate oligonucleotide (length) sequences of the amino acidsequences of two peptide fragments (SEQ ID NOS: 3 and 4) of the IKKα(see FIG. 1) were searched in the GenBank DNA sequence database. Thissearch revealed that nucleotide sequences encoding both peptidefragments were present in a partial cDNA encoding a portion of a proteindesignated human CHUK (GenBank Accession #U22512; Connelly and Marcu,supra, 1995).

Based on the human CHUK cDNA sequence, PCR primers were preparedcorresponding to the 5′-terminus (5′-CCCCATATGTACCAGCATCGGGAA-3′; SEQ IDNO: 5) and 3′-terminus (31-CCCCTCGAGTTCTGTTAACCAACT-5′; SEQ ID NO: 6).SEQ ID NO: 5 also contains a Nde I restriction endonuclease site(underlined) and an ATG (AUG) methionine codon (bold) and SEQ ID NO: 6also contains an Xho I site. RNA was isolated from HeLa cells and firststrand cDNA was prepared and used for a template by PCR using SEQ IDNOS: 5 and 6 as primers. The resulting 2.1 kilobase (kb) fragment wasgel purified, ³²P-labeled using oligo-dT and random primers, and used toscreen a human fetal brain library (Clontech; Palo Alto Calif.) underhigh stringency conditions (50% formamide, 42° C.; Sambrook et al.,supra, 1989).

In order to obtain the 5′-end of the cDNA encoding IKKβ, positiveplaques from above were screened by PCR using two internal primers,(5′-CATGGCACCATCGTTCTCTG-3′; SEQ ID NO: 7), which is complementary tothe sequence including the Ban I site around position 136 of SEQ ID NO:1, and (5′-CTCAAAGAGCTCTGGGGCCAGATAC-3′; SEQ ID NO: 8), which iscomplementary to the sequence including the Sac I site around position475, and a vector specific primer (TCCGAGATCTGGACGAGC-3′; SEQ ID NO: 9),which is complementary to vector sequences at the 5′-end of the cDNAinsert. The longest PCR product was selected and sequenced by thedideoxy method.

DNA sequencing revealed that the cloned IKKα cDNA contained anadditional 31 amino acids at the N-terminus as compared to human CHUK.The human IKKα shares a high amount of sequence identity with a proteindesignated mouse CHUK (GenBank Accession #U12473; Connelly and Marcu,supra, 1995). Although the mouse CHUK contains a domain havingcharacteristics of a serine-threonine protein kinase, no functionalactivity of the protein was reported and no potential substrates wereidentified. The putative serine-threonine protein kinase domain of humanCHUK was truncated at the N-terminus.

B. Expression of Human IKKα or of an Antisense IKKα Nucleic Acid in aCell:

The full length IKKα cDNA and a cDNA encoding the Δ31 human CHUK protein(Connelly and Marcu, supra, 1995) were subcloned into the Nde I and XhoI sites of a bacterial expression vector encoding a carboxy terminalFLAG epitope and HIS6 tag. Mammalian cell expression vectors wereconstructed by cleaving the bacterial expression vector with Nde I andHind III, to release the cDNA inserts, converting the ends of theinserts to blunt ends using Klenow polymerase, and ligating the cDNAinserts encoding the full length IKKα or the Δ31 human CHUK into pCDNA3(Invitrogen).

Alternatively, the IKKα cDNA and Δ31 cDNA were subcloned into the Bst XIsite of the pRcβactin vector (DiDonato et al., supra, 1996). Orientationof the inserts (sense or antisense) was determined by restrictionendonuclease mapping and partial sequence using vector-specific primers.Vector containing the cDNA's inserted in the sense orientation wereexamined for expression of the encoded product by immunoblot analysisusing an antibody specific for the FLAG epitope.

Transfection experiments were performed to determine the effect ofexpressing the cloned IKKα in HeLa cells or of expressing the clonedIKKα cDNA in the antisense orientation. One day prior to performing thetransfections, HeLa cells were split into 35 mm dishes to approximately50% confluency. Cells were transfected with 0.25 μg of a luciferasereporter gene containing an IL-8 promotor (Eckman et al., Amer. Soc.Clin. Invest. 96:1269-1279 (1995), which is incorporated herein byreference) along with either 1 μg pCDNA3 (Invitrogen, La Jolla Calif.;vector control), 1 μg pRcβactin-IKKα-AA (sense orientation), 1 μgpRcβactin-IKKα-K (antisense), or 0.1 μg pCDNA-IKKα-K using theLIPOFECTAMINE method as recommended by the manufacturer (GIBCO/BRL,Gaithersburg Md.). Total DNA concentrations were kept constant byaddition of empty pRcβactin DNA.

Transfected cells were incubated in DMEM containing 10% FBS for 24 hr.The cells then were washed and the growth medium was replaced with DMEMcontaining 0.1% FBS. Cells either were left untreated, or were treatedwith 20 ng/ml TNFα, 20 ng/ml IL-1α, or 100 ng/ml TPA (phorbol ester) for3.5 hr. Cells were harvested by scraping and washed once with PBS, thenlysed in 100 μl PBS containing 1% TRITON-X100. Luciferase assays wereperformed using 20 μl of lysate (DiDonato et al., supra, 1995). Theprotein concentration of each extract was determined using the BIORADprotein assay kit and luciferase activity was normalized according tothe protein concentrations.

NF-κB is known to induce expression for the IL-8 promotor. Thus, asexpected, treatment of the vector transfected control cells with TNFα,IL-1α or TPA resulted in a 3- to 5-fold increase in normalizedluciferase activity. In comparison, in cells transfected with the cDNAencoding IKKα, treatment with TNFα, IL-1α or TPA potentiated inductionof luciferase activity 5- to 6-fold above the level of inductionobserved in the vector transfected cells. These results indicate thatexpression of IKKα in cells increased the amount of NF-κB activated inresponse to the inducing agents.

In cells transfected with the vector expressing the antisense IKKαnucleic acid molecule, transcription of the luciferase reporter geneinduced by IL-1 or TNFα was at the limit of detection, indicatingtranscription was almost completely inhibited due to expression of theantisense IKKα. This result indicates that the native IKKα is turnedover relatively rapidly in the cells. Furthermore, treatment of thecells with the various inducing agents had no effect on the level ofluciferase expression of control reporter genes, which are notresponsive to NF-κB, as compared to the untreated cells. Otherappropriate control experiments were performed in parallel. Theseresults demonstrate the an expression of an antisense IKKα nucleic acidmolecule in a cell can specifically inhibit NF-κB mediated geneexpression.

EXAMPLE III Identification and Characterization of a Full Length HumanIKKβ Subunit

This example provides methods for isolating a nucleic acid moleculeencoding an IKKβ catalytic subunit of IKK and characterizing theactivity of the IKKβ subunit.

A. Cloning of IKKβ cDNA:

IKKβ was purified following SDS-PAGE and subjected to internal peptidesequencing (Example I). Five peptide sequences were obtained as follows:KIIDLGYAK (SEQ ID NO: 10); KXVHILN (M/Y) (V/G) (T/N/R/E/) (G/N) TI(H/I/S) (SEQ ID NO: 11; KXXIQQD (T/A) GIP (SEQ ID NO: 12); KXRVIYTQL(SEQ ID NO: 13); and KXEEVVSLMNEDEK (SEQ ID NO: 20), where amino acidresidues that could not be unambiguously determined are indicated by an“X” and where amino acids that could not be distinguished are shown inparentheses. These peptide sequences were used to screen the NCBI ESTdatabase and a 336 base pair EST (EST29518; Accession No. AA326115)encoding SEQ ID NOS: 13 and 20 was identified. This EST was determinedto correspond to amino acid residues 551 to 661 of SEQ ID NO: 15.

cDNA corresponding to the EST was obtained by PCR using first strandHeLa cDNA as a template and used to probe a human fetal brain library(Clontech). A 1 kb fragment was identified and used as a probe to screena plasmid based B cell library (Invitrogen). A 3 kb cDNA insert wasisolated and sequenced (FIG. 2; SEQ ID NO: 14) and encoded the fulllength IKKβ (SEQ ID NO: 15), including all five proteolytic fragments(see FIG. 3).

Comparison of the amino acid sequences of IKKα and IKKβ revealed greaterthan 50% amino acid identity (FIG. 3). In addition, SEQ ID NO: 15contains a kinase domain, which shares 65% amino acid identity withIKKα, a leucine zipper and a helix-loop-helix domain. Based on thesequence homology and domain structure, the polypeptide (SEQ ID NO: 15)was determined to be a member of the IKK catalytic subunit family ofproteins with IKKα and, therefore, was designated IKKβ.

B. Characterization of IKKβ:

This section describes the results of various assays characterizing IKKβactivity, particularly with regard to its association with IKKα. Inaddition, northern blot analysis revealed that IKKβ and IKKα arecoexpressed in most tissues examined, including pancreas, kidney,skeletal muscle, lung, placenta, brain, heart, peripheral bloodlymphocytes, colon, small intestine, prostate, thymus and spleen.

1. IKKβ Kinase Activity

The kinase activity associated with IKKβ was characterized using HeLa or293 cells transiently transfected with an HA-tagged IKKβ expressionvector. Transfected cells were stimulated with 20 ng/ml TNF for 10 minand HA-IKKβ was isolated by immunoprecipitation using anti-HA antibody(Kolodziej and Young, Meth. Enzymol. 194:508-519 (1991)). The immunecomplexes were tested for the ability to phosphorylate wild type (wt)and mutant forms of IκBα and IκBβ (see Example I).

Similarly to the purified IKK complex and the complex associated withIKKα, the IKKβ immune complex phosphorylated wt IκBα and IκBβ, but notmutants in which the inducible phosphorylation sites (Ser-32 and Ser-36for IκBα and Ser-19 and Ser-23 for IκBα) were replaced with eitheralanines or threonines. However, a low level of residual phosphorylationof full length IκBα (A32/A36) was observed due to phosphorylation ofsites in the C-terminal portion of the protein (DiDonato et al., supra,1997). Single substitution mutants, IκBα (A32) and IκB(A36), werephosphorylated almost as efficiently as wt IκBα, indicating thatIKKβ-associated IKK activity can phosphorylate IκBα at both Ser-32 andSer-36.

The response of IKKβ-associated kinase activity to various stimuli alsowas examined in HeLa cells transiently transfected with the HA-IKKβexpression vector. After 24 hr. the cells were stimulated with either 10ng/ml IL-1, 20 ng/ml TNF or 100 ng/ml TPA, then HA-IKKβ immune complexeswere isolated by immunoprecipitation and IKK activity was measured. TNFand IL-1 potently stimulated IKKβ-associated kinase activity, whereasthe response to TPA was weaker. The kinetics of IKKβ activation byeither TNF or IL-1 essentially were identical to the kinetics ofactivation of the IKKα-associated IκB kinase measured by a similarprotocol.

2. Functional Interactions Between IKKα and IKKβ

As shown in Example I, IKKα and IKKβ copurified in about a 1:1 ratiothrough several chromatographic steps, suggesting that the two proteinsinteract with each other. The ability of the IKK subunits to interact ina functional complex and the effect of each subunit on the activity ofthe other subunit was examined using 293 cells transfected withexpression vectors encoding Flag(M2)-IKKα or M2-IKKα and HA-IKKβ, eitheralone or in combination (see Hopp et al., BioTechnology 6:1204-1210(1988)). After 24 hr, samples of the cells were stimulated with TNF,lysates were prepared from stimulated and unstimulated cells, and oneportion of the lysates was precipitated with anti-Flag antibodies(Eastman Kodak Co.; New Haven Conn.) and another portion wasprecipitated with anti-HA antibodies. The IKK activity associated withthe different immune complexes and their content of IKKα and IKKβ weremeasured.

Considerably more basal IKK activity was precipitated with HA-IKKβ thanwith Flag-IKKα. However, the activity associated with HA-IKKβ wasfurther elevated upon coexpression of M2-IKKα and the low basal activityassociated with Flag-IKKα was strongly augmented by coexpression ofIKKβ. Immunoblot analysis revealed that the potentiating effect of suchcoexpression was not due to changes in the level of expression of IKKαor IKKβ.

The levels of IKK activities associated with IKKα and IKKβ were comparedmore precisely by transfecting 293 cells with increasing amounts ofHA-IKKα or HA-IKKβ expression vectors (0.1 to 0.5 μg/10⁶ cells) anddetermining the kinase activities associated with the two proteins incell lysates prepared before or after TNF stimulation (20 ng/ml, 5 min);GST-IκBα (1-54) was used as substrate. The level of expression of eachprotein was determined by immunoblot analysis and used to calculate therelative levels of specific IKK activity.

The HA-IKKα-associated IKK had a low level of basal specific activity,whereas expression of HA-IKKβ resulted in high basal specific activitythat was increased when higher amounts of HA-IKKβ were expressed.However, the specific IKK activity associated with either IKKα or IKKβisolated from TNF-stimulated cells was very similar and was notconsiderably affected by their expression level. These results indicatethat titration of a negative regulator or formation of a constitutivelyactive IKK complex can occur due to overexpression of IKKβ.

The ability of IKKα and IKKβ to physically interact was examined.Immunoblot analysis demonstrated that precipitation of HA-IKKβ using ananti-HA antibody coprecipitated both endogenous IKKα and coexpressedFlag-IKKα, as indicated by the higher amount of coprecipitating IKKαdetected after cotransfection with Flag-IKKα. Similarly,immunoprecipitation of Flag-IKKα with anti-Flag(M2) antibody resulted incoprecipitation of cotransfected HA-IKKβ. Exposure of the cells to TNFhad no significant effect on the association of IKKα and IKKβ.

The interaction between IKKα and IKKβ was further examined bytransfecting HeLa cells with various amounts (0.1 to 1.0 μg/10⁶ cells)of the HA-IKKβ vector. After 24 hr, the cells were incubated for 5 minin the absence or presence of 20 ng/ml TNF, then lysed. The lysates wereexamined for IKK activity and for the amount of HA-IKKβ and endogenousIKKα. Expression of increasing amounts of HA-IKKβ resulted in higherbasal levels of IKK activity and increasing amounts of coprecipitatedIKKα. The level of TNF stimulated IKK activity increased only marginallyin response to IKKβ overexpression and TNF had no effect on theassociation of IKKβ and IKKα.

Since the results described above revealed that HA-IKKβ associates withendogenous IKKα to generate a functional cytokine-regulated IKK complex,this association was examined further by transfecting HeLa cells witheither empty expression vector or small amounts (1 μg/60 mm plate) ofeither HA-IKKα or HA-IKKβ vectors. After 24 hr, samples of thetransfected cell populations were stimulated with 20 ng/ml TNF for 5min, then cell lysates were prepared and separated by gel filtration ona SUPEROSE 6 column. One portion of each column fraction wasimmunoprecipitated with a polyclonal antibody specific for IKKα andassayed for IKKα-associated IKK activity, while a second portion wasprecipitated with anti-HA antibody and examined for HA-IKKβ- orHA-IKKα-associated IKK activity. Relative specific activity wasdetermined by immunoprecipitating the complexes, separating the proteinsby SDS-PAGE, blotting the proteins onto IMOBILON membranes (Millipore;Bedford Mass.), immunoblotting with anti-HA antibody and quantitatingthe levels of IκB phosphorylation and HA-tagged proteins byphosphoimaging. The results demonstrated that endogenous IKKα-associatedIKK activity exists as two complexes, a larger complex of approximately900 kDa and a smaller one of approximately 300 kDa. Stimulation with TNFincreased the IKK activity of both complexes, although the extent ofincrease was considerably greater for the 900 kDa complex.

HA-IKKβ-associated IKK activity had exactly the same distribution as theIKKα-associated activity, eluting at 900 kDa and 300 kDa and, again, theextent of TNF responsiveness was considerably greater for the 900 kDacomplex. Comparison to the IKKα-associated activity in cells transfectedwith the empty vector indicated that HA-IKKβ expression produced amodest, approximately 2-fold increase in the relative amount of IKKactivity associated with the smaller 300 kDa complex. These resultsindicate that the 300 kDa IKK complex, like the 900 kDa complex,contains both IKKα and IKKβ. However, the 300 kDa lacks other subunitspresent in the 900 kDa complex. When IKKβ was overexpressed, therelative amount of the smaller complex increased, indicating that someof the subunits that are unique to the larger complex are present in alimited amount.

3. Both IKKα and IKKβ Contribute to IKK Activity

The relative contribution of IKKα and IKKβ to IKK activity was examinedby constructing mutant subunits in which the lysine (K) codon present atposition 44 of each subunit was substituted with a codon for eithermethionine (M) or alanine (A) codon, respectively. Similar mutations inother protein kinases render the enzymes defective in binding ATP and,therefore, catalytically inactive (Taylor et al., Ann. Rev. Cell Biol.8:429-462 (1992)). The activity of the IKK mutants was compared to theactivity of their wild type (wt) counterparts by cell-free translationin reticulocyte lysates using GST-IκBα (1-54) as a substrate.Translation of IKKα(KM) resulted in formation of IκB kinase having onlyslightly less activity than the IKK formed by translation of wt IKKα. Incomparison, translation of IKKβ(KA) did not generate IKK activity.Translation of wt IKKβ generated IκB kinase activity as expected.

The activities of the different proteins also was examined by transienttransfection in mammalian cells. Expression and immunoprecipitation ofHA-IKKα(KM) resulted in isolation of cytokine stimulated IKK activitythat, after TNF stimulation, was 2-to 3-fold lower than the activity ofIKK formed by wt HA-IKKα isolated from TNF-stimulated cells. Similarly,expression and immunoprecipitation of HA-IKKβ resulted in formation of acytokine responsive IKK activity that, after TNF stimulation, was 3- to5-fold lower than the activity of IKK generated by wt HA-IKKβ isolatedfrom TNF stimulated cells. In contrast to results obtained byoverexpression of wt HA-IKKβ, however, overexpression of HA-IKKβ(KA) didnot result in the generation of basal IKK activity. Immunoprecipitationexperiments revealed that IKKα(KM) associates IKKβ and that IKKβ(KA)associates with IKKα and that both IKKα and IKKβ undergo homotypicinteractions as efficiently as they undergo heterotypic interactions.

Autophosphorylation of wt and kinase-defective HA-IKKα and HA-IKKβ wasexamined in transiently transfected HeLa cells. HeLa cells expressingthese proteins were treated with TNF for 10 min, then cell lysates ofTNF treated or untreated cells were immunoprecipitated with HAantibodies and the immune complexes were subjected to a phosphorylationreaction (DiDonato et al., supra, 1997). Both wt HA-IKKα and wt HA-IKKβwere phosphorylated and their autophosphorylation was enhanced inTNF-stimulated extracts. In contrast, the kinase-defective IKKα or IKKβmutants did not exhibit significant autophosphorylation.

4. The Role of the LZ and HLH Motifs in IKKα and IKKβ

IKKα and IKK, both contain leucine zipper (LZ) and helix-loop-helix(HLH) motifs, which are known to mediate protein-protein interactionsthrough their hydrophobic surfaces. The role of the LZ motif in the IKKsubunit interaction was examined using an IKKα mutant in which the L462and L469 residues within the LZ region were substituted with serineresidues. The role of the HLH motif was examined using an HLH mutant ofIKKα containing a substitution of L605 with arginine (R) and of F606with proline (P). The activity of the IKKα LZ⁻ and HLH⁻ mutants wasexamined by transient transfection in 293 cells, either alone or in thepresence of cotransfected Flag-IKKα.

Expression of wt HA-IKKα generated substantial IKK activity that wasisolated by immunoprecipitation with anti-HA, whereas very little IKKactivity was generated in cells transfected with either the HA-IKKα(LZ)⁻or HA-IKKα(HLH)⁻ mutant. Coexpression of the mutant IKK subunits withFlag-IKKβ resulted in a substantial increase in the IKK activityisolated by immunoprecipitation of HA-IKKα, but had no effect on thevery low activity that coprecipitated with HA-IKKα(LZ)⁻. However,coexpression of Flag-IKKβ did stimulate the low level of IKK activityassociated with HA-IKKα(HLH)⁻. Probing of the HA immune complexes withanti-Flag(M2) antibodies indicated that both wt HA-IKKα andHA-IKKα(HLH)⁻ associated with similar amounts of Flag-IKKβ, but that theHA-IKKα(LZ)⁻ mutant did not associate with Flag-IKKβ. These resultsindicate that the lower IκB kinase activity associated with theIKKα(LZ)⁻ mutant is due to a defect in its ability to interact withIKKβ. The lower IκB kinase activity of the IKKα(HLH)⁻ mutant, on theother hand, likely is due to a defect in the ability to interact with asecond, undefined protein, since the HLH mutant can interact with IKKβ.

5. Both IKKα and IKKβ are Necessary for NF-κB Activation

The contribution of IKKα and IKKβ to NF-κB activation was examined usingHeLa cells transfected with expression vectors encoding HA-tagged wtIKKα, IKKα(KM), wt IKKβ and IKKβ(KA); an HA-JNK1 vector was used as acontrol. NF-κB activation was assessed by examining the subcellulardistribution of RelA(p65) by indirect immunofluorescence.

HeLa cells were grown on glass cover slips in growth medium, thentransfected with 1 μg plasmid DNA by the lipofectamine method. After 24hr, samples of cells were stimulated with 20 ng/ml TNF for 30 min, thenstimulated or unstimulated cells were washed with PBS and fixed with3.5% formaldehyde in PBS for 15 min at room temperature (RT). The fixedcells were permeablized with 0.02% NP-40 in PBS for 1 min, thenincubated with 100% goat serum at 4° C. for 12 hr. The cells then werewashed 3 times with PBS and incubated with a mixture of a rabbitanti-NF-κB p65 (RelA) antibody (1:100 dilution; Santa Cruz Biotech) anda mouse monoclonal anti-HA antibody in PBS containing 1% BSA and 0.2%TRITON X-100 at 37° C. for 2 hr. Cells then were washed 3 times with PBScontaining 0.2% TRITON X-100 and incubated for 2 hr at RT with secondaryantibodies, fluorescein-conjugated goat affinity purified anti-mouseIgG-IgM and rhodamine-conjugated IgG fraction goat anti-rabbit IgG(1:200 dilution; Cappel). Cells were washed 4 times with PBS containing0.2% TRITON X-100, then covered with a drop of gelvatol mountingsolution and viewed and photographed using a Zeiss Axioplan microscopeequipped for epifluorescence with the aid of fluoroscein and rhodaminespecific filters.

Double staining with both anti-RelA and anti-HA revealed that expressionof moderate amounts of either wt IKKα or wt IKKβ did not produceconsiderable stimulation of RelA nuclear translocation. In addition, thewt IKK proteins did not interfere with the nuclear translocation of RelAinduced by TNF treatment. However, expression of similar levels ofeither IKKα(KM) or IKKβ(KA), as determined by the intensity of thefluorescent signal, inhibited the nuclear translocation of RelA inTNF-treated cells. Expression of HA-JNK1 had no effect on thesubcellular distribution of RelA. Since the subcellular distribution ofRelA is dependent on the state and abundance of IκB, these resultsindicate that expression of either IKKα(KM) or IKKβ(KA) inhibits theinduction of IκB phosphorylation and degradation by TNF.

EXAMPLE IV Isolation of IκB Kinase Complex

This example demonstrates a method for isolating the 900 kDa IκB kinasecomplex comprising an IKKα polypeptide.

Proteins that associate with IKKα in vivo were isolated byimmunoprecipitation using HIS6 and FLAG epitope tags. The HIS6-FLAG-IKKα(HF-IKKα) encoding construct was prepared using a double strandedoligonucleotide, 5′-AGCTTGCGCGTATGGCTTCGGGTCATCACCATCACCATCACGGTGACTACAAGGACGACGATGACAAAGGTGACATCGAAGGTAGAGGTCA-3′ (SEQ ID NO:16), which encodes six histidine residues (HIS6), the FLAG epitope andthe factor Xa site in tandem. The oligonucleotide was inserted usingHindIII-NdeI site in frame with the N-terminus of the IKKα codingsequence in the BLUESCRIPT KS plasmid (Stratagene; La Jolla Calif.). TheHindIII-NotI fragment of this plasmid, which contains the HF-IKKα cDNAsequence, was subcloned into the pRcβactin mammalian expression vector,which contains a nucleic acid sequence conferring neomycin resistance,to produce plasmid pRC-HF-IKKα. Expression of the HF-IKKα polypeptidewas confirmed by western blot analysis using anti-FLAG antibodies.

pRC-HF-IKKα was transfected into human embryonic kidney 293 cells andtransfected cells were selected for growth in the presence of G418. Alow basal level of IKK activity was detected in cells expressing HF-IKKαand IKK activity increased several fold when the cells were treated withTNFα. This result indicates that the HF-IKKα expression in 293 cells isassociated with IKK activity in the cells and that such IKK activity isinducible in response to TNFα.

A 293 cell line that expresses HF-IKKα was selected and expanded toapproximately 4×10⁸ cells. The cells were treated with 10 ng/ml TNFα for5 min, then harvested in ice cold PBS by centrifugation at 2500×g. Thecell pellet was washed with ice cold PBS, resuspended in lysis buffer(20 mM Tris, pH 7.6), 150 mM NaCl, 1% TRITON X-100, 20 mMβ-glycerophosphate, 2 mM PNPP, 1 mM Na₃VO₄, 5 mM β-mercaptoethanol, 1 mMEDTA, 0.5 mM EGTA, 1 mM PMSF, 3 μg/ml pepstatin, 3 μg/ml leupeptin, 10μg/ml bestatin and 25 μg/ml aprotinin), and lysed by 20 strokes in aglass Dounce homogenizer (pestle A).

The homogenate was centrifuged at 15,000 rpm in a Beckman SS34 rotor for30 min at 4° C. The supernatant was collected, supplemented with 20 mMimidazole and 300 mM NaCl, then mixed with 0.5 ml of a 50% slurry ofNi-NTA (nickel nitrilotriacetic acid; Qiagen, Inc.; Chatsworth Calif.)and stirred for 4 hr at 4° C. Following incubation, the resin waspelleted at 200×g and the supernatant was removed. The resin was washed3 times with 50 ml binding buffer containing 25 mM imidazole.

Proteins bound to the resin were eluted in 2 ml binding buffercontaining 150 mM imidazole and 20 mM DTT. The eluate was mixed with 100μl of a 50% slurry of anti-FLAG antibody coupled to SEPHAROSE resinusing the AMINOLINK PLUS immobilization kit (Pierce Chem. Co.; RockfordIll.) and stirred for 4 hr at 4° C. The resin was pelleted at 1000×g,the supernatant was removed, and the resin was washed with 10 ml bindingbuffer (without imidazole). Proteins bound to the resin then were elutedwith 1 SDS or with FLAG peptide and examined by 10% SDS-PAGE.

Silver staining revealed the presence of seven proteins, including theHF-IKKα, which was confirmed by western blot analysis using anti-FLAGantibody. The copurified proteins had apparent molecular masses of about100 kDa, 63 kDa, 60 kDa, 55 kDa, 46 kDa and 29 kDa; the endogenous 87kDa IKKβ comigrates with the HA-IKKα protein. These results indicatethat IKKα, along with some or all of the copurifying proteins, comprisethe 900 kDa IκB kinase complex.

EXAMPLE V Anti-IKK Antisera

This example provides a method of producing anti-IKK antisera.

Anti-IKKα antibodies were raised in rabbits using either His-tagged IKKαexpressed in E. coli or the IKKα peptide ERPPGLRPGAGGPWE (SEQ ID NO: 17)or TIIHEAWEEQGNS (SEQ ID NO: 18) as an immunogen. Anti-IKKβ antibodieswere raised using the peptide SKVRGPVSGSPDS (SEQ ID NO: 19). Thepeptides were conjugated to keyhole limpet hemocyanin (Sigma ChemicalCo.; St. Louis Mo.). Rabbits were immunized with 250 to 500 μgconjugated peptide in complete Freund's adjuvant. Three weeks after theprimary immunization, booster immunizations were performed using 50 to100 μg immunogen and were repeated three times, at 3 to 4 weekintervals. Rabbits were bled one week after the final booster andantisera were collected. Anti-IKKα antiserum was specific for IKKα anddid not cross react with IKKβ.

EXAMPLE VI Use of an IKK Subunit in a Drug Screening Assay

This example describes an assay for screening for agents such as drugsthat alter the association of an IKK subunit and a second protein thatspecifically associates with the IKK subunit.

A GST-IKK subunit fusion protein or HIS6-IKK subunit fusion protein canbe prepared using methods as described above and purified usingglutathione- or metal-chelation chromatography, respectively (Smith andJohnson, Gene 67:31-40 (1988), which is incorporated herein byreference; see, also, Example IV). The fusion protein is immobilized toa solid support taking advantage of the ability of the GST protein tospecifically bind glutathione or of the HIS6 peptide region to chelate ametal ion such as nickel (Ni) ion or cobalt (Co) ion (Clontech) byimmobilized metal affinity chromatography. Alternatively, an anti-IKKantibody can be immobilized on a matrix and the IKK-α can be allowed tobind to the antibody.

The second protein, which can be IκB or a protein that copurifies withIKK subunit as part of the 900 kDa IκB kinase, for example, can bedetectably labeled with a moiety such as a fluorescent molecule or aradiolabel (Hermanson, supra, 1996), then contacted in solution with theimmobilized IKK subunit under conditions as described in Example I,which allow IκB to specifically associate with the IKK subunit.Preferably, the reactions are performed in 96 well plates, which allowautomated reading of the reactions. Various agents such as drugs thenare screened for the ability to alter the association of the IKK subunitand IκB.

The agent and labeled IκB, for example, can be added together to theimmobilized IKK subunit, incubated to allow binding, then washed toremove unbound labeled IκB. The relative amount of binding of labeledIκB in the absence as compared to the presence of the agent beingscreened is determined by detecting the amount of label remaining in theplate. Appropriate controls are performed to account, for example, fornonspecific binding of the labeled IκB to the matrix. Such a methodallows the identification of an agent that alter the association of anIKK subunit and a second protein such as IκB.

Alternatively, the labeled IκB or other appropriate second protein canbe added to the immobilized IKK subunit and allowed to associate, thenthe agent can be added. Such a method allows the identification ofagents that can induce the dissociation of a bound complex comprisingthe IKK subunit and IκB. Similarly, a screening assay of the inventioncan be performed using the 900 kDa IKK complex, comprising an IKKsubunit.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1. An isolated monoclonal antibody that specifically binds to thepeptide set forth in SEQ ID NO: 19 and to the protein set forth as SEQID NO:
 15. 2. An isolated cell line producing the antibody of claim 1.3. The cell line of claim 2, which is a hybridoma cell line.
 4. Anisolated antibody that specifically binds to the peptide set forth inSEQ ID NO: 19 and to the protein set forth as SEQ ID NO:
 15. 5. A methodfor isolating IκB kinase subunit (IKKβ) from a sample, comprising: a)providing: i) antibody of claim 4; and ii) a sample; b) contacting saidantibody with said sample under conditions for binding said antibodywith IKKβ; and c) isolating IKKβ bound to said antibody.
 6. The methodof claim 5, wherein said IKKβ is comprised in IκB kinase.
 7. A methodfor detecting IκB kinase subunit (IKKβ) in a sample, comprising: a)providing: i) antibody of claim 4; and ii) a sample; b) contacting saidantibody with said sample under conditions for binding said antibodywith IKKβ; and c) detecting IKKβ bound to said antibody.
 8. The methodof claim 7, further comprising determining the level of IKKβ bound tosaid antibody.
 9. The method of claim 7, wherein said IKKβ is comprisedin IκB kinase.